Short fatty acid tail polymyxin derivatives and uses thereof

ABSTRACT

The present invention relates to a polymyxin derivative wherein the derivative has a total of three positive charges at physiological pH and wherein the terminal moiety (D) of the derivative comprises a total of 1 to 5 carbon atoms. The invention also relates to a method of treating a subject for a gram-negative bacterial infection by administering a polymyxin derivative of the invention in combination with a second antibacterial agent. Finally, the invention relates to a process for preparing such polymyxin derivatives.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/366,213, filed Feb. 5, 2009, which claims priority to U.S.Provisional Application No. 61/065,214, filed Feb. 8, 2008, and U.S.Provisional Application No. 61/127,933, filed May 16, 2008, the entirecontents of which are hereby incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to polymyxin derivatives and to usesthereof in the treatment of infections caused by Gram-negative bacteria.The polymyxin derivatives of the present invention are especially usefulin sensitizing bacteria to enhance the effects of other antibacterialagents.

BACKGROUND

Sepsis kills more than 215,000 Americans each year. It is estimated that750,000 Americans are infected with severe sepsis and 29% of them diefrom it each year. Sepsis deaths make 9% of all death cases in the U.S.Sepsis kills as many Americans as myocardial infections, even more thantraffic accidents.

Two to three million Americans acquire a hospital infection each yearand 10% of these infections progress to sepsis. More than 90,000 ofthese patients die from sepsis infected in hospitals.

Severe sepsis and septic shock (severe sepsis combined with low bloodpressure) took up to 135,000 lives each year in the intensive care units(ICU) in the European Union according to the OECD Health Report of 2000.In Britain, 5,000 out of 100,000 patients who acquired a hospitalinfection die from sepsis every year in acute care hospitals belongingto the NHS organisation.

The death toll has increased year after year due to the fact that thenumber of patients predisposed to sepsis, such as the elderly, prematureneonates, and cancer patients, has increased, not least because manyserious illnesses are more treatable than before. Also the use ofinvasive medical devices and aggressive procedures has increased.

Gram-negative bacteria cause more than 40% of all septicemic infectionsand many of the Gram-negative bacteria are extremely multiresistant.Gram-negative bacteria provide a harder challenge in therapy thanGram-positives, as they possess a unique structure, the outer membrane,as their outermost structure. Lipopolysaccharide molecules located onthe outer membrane inhibit the diffusion of many antibacterial agentsdeeper into the cell, where their ultimate targets are located. Morethan 95% of the novel antibacterial agents isolated from nature orchemically synthesized in 1972-1991 lacked activity againstGram-negatives (Vaara 1993).

Polymyxins are a group of closely related antibiotic substances producedby strains of Paenibacillus polymyxa and related organisms. Thesecationic drugs are relatively simple peptides with molecular weights ofabout 1000. Polymyxins, such as polymyxin B, are decapeptideantibiotics, i.e., they are made of ten (10) aminoacyl residues. Theyare bactericidal and especially effective against Gram-negative bacteriasuch as Escherichia coli and other species of Enterobacteriaceae,Pseudomonas, Acinetobacter baumannii, and others. However, polymyxinshave severe adverse effects, including nephrotoxicity and neurotoxicity.These drugs thus have limited use as therapeutic agents because of highsystemic toxicity.

Polymyxins have been used in the therapy of serious infections caused bythose bacteria, but because of the toxicity, their use was largelyabandoned in the 70's when newer, better tolerated antibiotics weredeveloped. The recent emergence of multiresistant strains ofGram-negative bacteria has necessitated the therapeutic use ofpolymyxins as the last resort, in spite of their toxicity, and as manyof the less toxic antibiotics have already lost their effectivenessagainst particular strains of the said bacteria, the use of polymyxinshas again increased.

Accordingly, polymyxins have now been recalled to the therapeuticarsenal, although, due to their toxicity, on a very limited scale. Theirsystemic (i.e., non-topical) use is, however, largely restricted to thetherapy of life-threatening infections caused by multiply resistantstrains of Ps. aeruginosa and A. baumannii as well as bycarbapenem-resistant enteric bacteria.

Polymyxins consist of a cyclic heptapeptide part and a linear partconsisting of a tripeptide portion and a hydrophobic fatty acid taillinked to the α-amino group of the N-terminal amino acid residue of thetripeptide and may be represented by the general formula:

wherein R1-R3 represent the tripeptide side chain portion; R4-R10 theheptapeptide ring portion and R(FA) represents the hydrophobic fattyacid tail linked to the α-amino group of the N-terminal amino acidresidue of the tripeptide.

The polymyxin group includes the following polymyxins: A1, A2, B1-B6,IL-polymyxin B1, C, D1, D2, E1, E2, F, K1, K2, M, P1, P2, S, and T(Storm et al. 1977; Srinivasa and Ramachandran 1979). All polymyxins arepolycationic and possess five (5) positive charges, with the exceptionof polymyxin D, F, and S which possess four (4) positive charges. Itshould be noted that modified polymyxins that lack the fatty acid partR(FA) but carry R1-R10 have one additional positive charge when comparedto the natural polymyxins they derived from, due to the free a-aminogroup in the N-terminus of the derivative. Accordingly, for example,such a derivative of polymyxin B or polymyxin E carries six (6) positivecharges in total.

The clinically used polymyxin B and polymyxin E differ from each otheronly in the residue R6, which is D-phenylalanyl residue in polymyxin Band D-leucyl residue in polymyxin E.

Also circulin A and B are classified as polymyxins (Storm et al. 1977).They differ from other polymyxins only in carrying isoleucyl residue inthe position R7 whereas other polymyxins have either threonyl or leucylresidue in the said position. For an overview of the structures of somepolymyxins, see Table 1.

TABLE 1 The structure of selected polymyxins and octapeptin as well asselected derivatives thereof Compound R(FA) R1 R2 R3 R4 R5 R6 R7 R8 R9R10 SEQ ID NO: Polymyxin B MO(H)A- Dab- Thr- Dab- *Dab- Dab- D Phe- Leu-Dab Dab *Thr 1 Colistin (polymyxin E) MO(H)A- Dab- Thr- Dab- *Dab- Dab-D Leu- Leu- Dab Dab *Thr 17 Colistin sulphomethate MO(H)A- sm-Dab- Thr-sm-Dab- *Dab- Sm-Dab- D Leu- Leu- sm--Dab- sm--Dab- *Thr 18 Polymyxin AMO(H)A- Dab- Thr- D Dab- *Dab- Dab- D Leu- Thr- Dab Dab *Thr 19Polymyxin M MOA Dab- Thr- Dab- *Dab- Dab- D Leu- Thr- Dab Dab *Thr 20Polymyxin D MO(H)A- Dab- Thr- D-Ser- *Dab- Dab- D Leu- Thr- Dab Dab *Thr21 Circulin A MOA Dab- Thr- Dab- *Dab- Dab- D Leu- Ile- Dab Dab *Thr 22Octapeptin A OHMDA — — Dab- *Dab- Dab- D Leu- Leu- Dab Dab *Thr 23Deacylcolistin (DAC) Dab- Thr- Dab- *Dab- Dab- D Leu- Leu- Dab Dab *Thr24 Polymyxin E nonapeptide Thr- Dab- *Dab- Dab- D-Leu- Leu- Dab Dab *Thr25 (PMEN) Deacylpolymyxin B Dab- Thr- Dab- *Dab- Dab- D Phe- Leu- DabDab *Thr 26 (DAPB) Polymyxin B nonapeptide Thr- Dab- *Dab- Dab- D Phe-Leu- Dab Dab *Thr 4 (PMBN) Polymyxin B octapeptide Dab- *Dab- Dab- DPhe- Leu- Dab Dab *Thr 27 (PMBO) Polymyxin B heptapeptide *Dab- Dab- DPhe- Leu- Dab Dab *Thr 5 (PMHP)

Polymyxin B is represented by the following formula:

Commercially available polymyxin B is a mixture, where R—FA ispredominantly 6-methyloctanoyl (6-MOA, in polymyxin B1) but may also bea related fatty acyl such as 6-methylheptanoyl (6-MHA, in polymyxin B2),octanoyl (in polymyxin B3), or heptanoyl (polymyxin B4) (Sakura et al.2004). All these variants are equally potent against Gram-negatives suchas E. coli (Sakura et al. 2004). Quite analogously, in polymyxin E1(colistin A) and in circulin A the R—FA is 6-MOA and in polymyxin E2(colistin B) and in circulin B the R—FA is 6-MHA. Numerous researchershave attached various hydrophobic moieties including various fatty acylresidues to the N-terminus of polymyxin derivatives and analogues andhave shown that the resulting derivatives have potent antibacterialactivity (Chihara et al. 1973, Sakura et al. 2004 and in US patentpublication 2006004185. Even the derivative that carries the bulkyhydrophobic 9-fluorenylmethoxycarbonyl residue as the R—FA is almost aspotent as polymyxin B in inhibiting the growth of E. coli and otherGram-negative bacteria (Tsubery et al. 2001).

For biological activity the heptapeptide ring structure is essential(Storm et al. 1997). A derivative with an octapeptide ring issignificantly less active as an antibiotic.

Multiple modifications of polymyxins and multiple polymyxin-likesynthetic molecules have been made, and with certain limits they havepreserved their biological activity. The modifications comprise but arenot limited to those in the side chain, as well as molecules in which aninherent hydrophobic amino acid residue (such as DPhe or Leu) has beenreplaced with another hydrophobic amino acid residue or in which thecationic Dab has been replaced with another cationic amino acyl residue,such as Lys, Arg, or ornithine residue (Storm et al. 1997, Tsubery etal. 2000a, Tsubery et al. 2002, US patent publication 2004082505, Sakuraet al. 2004, US patent publication 2006004185).

Other modifications that result in microbiologically at least partiallyactive compounds comprise but are not limited to alkanoyl esters wherethe OH-groups of the threonyl residues form esters with alkanoyls suchas propionyl and butyryl (U.S. Pat. No. 3,450,687).

Octapeptins are otherwise identical to polymyxins but have a covalentbond instead of the residues R1-R2 (Table 1). In this invention, the Rpositions are numbered according to those in the natural polymyxins andthus the only amino acyl residue in the side chain of octapeptins isdefined as R3. Accordingly, octapeptins are octapeptides whereas allnatural polymyxins are decapeptides, and they possess only four (4)positive charges. The R—FA residues among various octapeptins (A1, A2,A3, B1, B2, B3, C1) include the following: 3-OH-8-methyldecanoic acid,3-OH-8-methylnonanoic acid, and β-OH-6-methyloctanoic acid. Derivativesthat possess a fatty acyl residue with 6 to 18 carbon atoms have apotent antibacterial activity against E. coli (Storm et al. 1977).

The first target of polymyxins in Gram-negative bacteria is their outermembrane (OM) that is an effective permeability barrier against manynoxious agents including large (Mw more than 700 d) antibiotics as wellas hydrophobic antibiotics. By binding to the lipopolysaccharide (LPS)molecules exposed on the outer surface of the OM, polymyxins damage thestructure and function of the OM and, as a result, permeabilize (i.e.,make permeable) the OM to polymyxin itself, as well as to many othernoxious agents (Nikaido and Vaara 1985, Vaara 1992, Nikaido 2003). Thefinal and lethal target (the bactericidal target) of polymyxins isbelieved to be the cytoplasmic membrane (the inner membrane) ofbacteria.

Numerous efforts have been made to reduce the toxicity of polymyxins.The treatment of polymyxin E (colistin) with formaldehyde and sodiumbisulfite yields colistin sulphomethate, in which the free amino groupsof the five diaminobutyric acid residues have partially been substitutedby sulphomethyl groups (Table 1). The preparations consist of undefinedmixtures of the mono-, di-, tri-, tetra-, and penta-substitutedcompounds. The sulphomethylated preparations, when freshly dissolved inwater, initially lack both the antibacterial activity and toxicity ofthe parent molecule, but when the compounds start decomposing in thesolution, in the blood or in the tissues to yield less substitutedderivatives and free colistin, both the antibacterial activity and thetoxicity are partially brought back. Furthermore, the degree of initialsulphomethylation apparently varies between the commercially availablepharmaceutical preparations. Many other ways to block all the free aminogroups have been published. Examples comprise but are not limited to theformation of unstable Schiff bases with amino acids (Storm et al. 1977).

Polymyxin E nonapeptide (PMEN, colistin nonapeptide, Table 1), obtainedby treating polymyxin E enzymatically and lacking the R—FA and R1, wasshown in 1973 to be less toxic than the parent compound in acutetoxicity assay (immediate death presumably due to direct neuromuscularblockade) in mice (Chihara et al. 1973). However, it also lacked theantibacterial activity, as measured as its ability to inhibit bacterialgrowth (Chirara et al. 1973). The role of the linear part may contributeto the antibacterial activity of the polymyxins.

Vaara and Vaara, on the other hand, showed, that polymyxin B nonapeptide(PMBN, Table 1) retains the ability to permeabilize the OM ofGram-negative bacteria (Vaara and Vaara 1983a,b,c; U.S. Pat. No.4,510,132; Vaara 1992). Accordingly, even though it lacks the directantibacterial activity (i.e., the ability to inhibit bacterial growth),it is able to sensitize (i.e., make sensitive or, as also termed, makesusceptible) the bacteria to many antibacterial agents such ashydrophobic antibiotics as well as large antibiotics and some othernoxious agents.

PMBN also sensitizes bacteria to the bactericidal activity of the humancomplement system, present in fresh human serum as a first-line defencesystem against invaders (Vaara and Vaara 1983a, Vaara et al. 1984, Vaara1992). Furthermore, it sensitizes the bacteria to the joint bactericidalactivity of serum complement and human polymorphonuclear white cells(Rose et al. 1999).

PMBN resembles PMEN in being less toxic in the acute toxicity assay inmice than unmodified polymyxins. In further toxicological assays,several criteria proved PBMN to be less toxic than its parent compound,but this polymyxin derivative was still judged to be too nephrotoxic forclinical use (Vaara 1992).

PMBN carries five (5) positive charges. Subsequent studies revealed,quite expectedly, that PMEN, also carrying five (5) positive charges aswell as deacylpolymyxin B and deacylpolymyxin E, both carrying six (6)positive charges are potent agents to sensitize bacteria to otherantibiotics (Viljanen et al. 1991, Vaara 1992). In addition, it has beenshown that a structurally further reduced derivative polymyxin Boctapeptide (PMBO) retains a very effective permeabilizing activitywhile polymyxin B heptapeptide (PMBH) is less active (Kimura et al.1992). PMBN, PMEN and PMBO have five (5) positive charges while PMBH hasonly four (4) positive charges. This difference may explain the weakeractivity of PMBH.

The group of Ofek, Tsubery and Friedkin recently describedpolymyxin-like peptides that were linked to chemotactic peptides, suchas fMLF, that attract polymorphonuclear leucocytes (US patentpublication 2004082505, Tsubery et al. 2005). They described peptidesfMLF-PMBN, MLF-PMBN, fMLF-PMEN, fMLF-PMBO and MLF-PMBO, all carryingfour (4) positive charges, that sensitize Gram-negative bacteria toantibiotics, even though no comparative studies with increasingconcentrations of the compounds were published (Tsubery et al. 2005).

In order to study the structures and functional properties ofpolymyxins, a few works have disclosed, among other compounds, polymyxinderivatives having less than four (4) positive charges.

Teuber (1970) has described the treatment of polymyxin B with aceticanhydride that yields a preparation containing polymyxin B as well asits mono-, di-, tri-, tetra-, and penta-N-acetylated forms. Teuber alsoseparated each group and nonquantitatively reported using an agardiffusion assay that penta-acetylated and tetra-acetylated forms lackedthe ability to halt the growth of Salmonella typhimurium, whereas di-and monoacetylated forms did have such ability. Triacetylated form hadsome ability.

Srinivasa and Ramachandran (1978) isolated partially formylatedpolymyxin B derivatives and showed that a diformyl derivative as well asa tri-formyl derivative inhibited the growth of Pseudomonas aeruginosa.They did not disclose the compounds' ability to sensitize bacteria toantibiotics. Furthermore, in 1980 they showed that the free amino groupsof triformyl-polymyxin B in residues R1 and R3, as well as the freeamino groups of diformylpolymyxin B in residues R1, R3, and R5 areessential while the free amino groups in R8 and R9 are not essential forthe growth inhibition (Srinivasa and Ramachandran, 1980a).

A shortened polymyxin B derivative octanoyl polymyxin B heptapeptide hasbeen disclosed by Sakura et al. (2004). The attachment of the octanoylresidue to the N-terminus of the residue R4 of the polymyxin Bheptapeptide results in a compound having only three (3) positivecharges. Sakura et al. found that octanoyl polymyxin B heptapeptideinhibits the growth of bacteria only at a very high concentration (128μg/ml), whereas the other derivatives such as octanoyl polymyxin Boctapeptide and octanoyl polymyxin B nonapeptide, both having fourcharges (4) were very potent agents to inhibit bacterial growth.

US patent publication 2006004185 recently disclosed certain polymyxinderivatives and intermediates that can be used to synthesize new peptideantibiotics. The antibacterial compounds described possessed four (4) orfive (5) positive charges.

Furthermore, closely related polymyxin B and polymyxin B₁ compounds havealso been disclosed by Okimura et al. (2007) and de Visser et al.(2003). Okimura et al. have studied the chemical conversion of naturalpolymyxin B and colistin to their N-terminal derivatives and de Visseret al. have studied solid-phase synthesis of polymyxin B₁ and analoguesvia a safety-catch approach. The antibacterial compounds described inthese works possessed four (4) or five (5) positive charges.

There is still an urgent need for polymyxin derivatives, which sensitizebacteria to enhance the effects of other antibacterial agents, foreffective treatments for bacterial infections, in particular for theinfections caused by multiresistant Gram-negative bacteria.

SUMMARY

The present invention relates to a polymyxin derivative wherein thetotal number of positive charges at physiological pH is three andwherein the derivative has a fatty acid tail (i.e., R(FA) or D)comprising 1 to 5 carbon atoms. It has been found that certain polymyxinderivatives of the invention having fatty acid tails of 1 to 5 carbonatoms may have improved pharmacokinetic properties as compared to nativepolymyxins, octapeptins, and polymyxin derivatives with longer fattyacid tails. Examples of these pharmacokinetic properties include, butare not limited to, longer serum half life, increased renal clearance,and/or increased urinary recovery.

The present invention pertains, at least in part, to polymyxinderivatives of formula (I):

wherein:

A is a polymyxin ring moiety;

D is a terminal moiety comprising 1 to 5 carbon atoms;

m¹, m², and m³ are each independently 0 or 1;

Q¹, Q², and Q³ are each independently CH₂, C═O, or C═S;

W¹, W², and W³ are each independently NR⁴, O, or S;

R^(1′), R^(2′), and R^(3′) are each independently side chains of naturalor unnatural amino acids, alkyl, alkenyl, alkyl, arylalkyl, aryl,alkoxy, alkoxycarbonyl, aryloxycarbonyl, alkylamino, or alkynyl; and

R⁴ is hydrogen or alkyl,

and pharmaceutically acceptable prodrugs and salts thereof, providedthat (1) when A is an octapeptin ring, m¹ and m² are 0, m³ is 1, W³ isNH, Q³ is C═O, and R^(3′) is the side chain of diaminobutyric acid(Dab), then D is not C₂-C₅ acyl, and (2) when D is acetyl, butanoyl orpentanoyl, then R^(3′) is not the side chain of Dab.

The invention is also directed to polymyxin derivatives of formula (II):

wherein:

A is a polymyxin ring moiety;

D is R¹²—C(′O), R¹²—C(═S), or R^(12′);

m¹, m², and m³ are each independently 0 or 1, provided that at least oneof m¹, m², and m³ are 1;

R^(1′), R^(2′), and R^(3′) are each independently side chains of naturalor unnatural amino acids, alkyl, alkenyl, arylalkyl, aryl, alkoxy,alkoxycarbonyl, aryloxycarbonyl, alkylamino, or alkynyl; and

R¹² is C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl,

R^(12′) is C₁-C₅ alkyl, C₂-C₅ alkenyl, or C₂-C₅ alkynyl,

and pharmaceutically acceptable prodrugs and salts thereof, providedthat (1) when A is an octapeptin ring, m¹ and m² are 0, m³ is 1, andR^(3′) is the side chain of diaminobutyric acid (Dab), and D is R¹²—C═O,then R¹² is not C₁-C₅ alkyl, and (2) when D is acetyl, butanoyl orpentanoyl, then R^(3′) is not the side chain of Dab.

In another embodiment, the invention also includes polymyxin derivativesof formula (III):

wherein:

A is a polymyxin B or polymyxin E ring moiety;

D is R¹²—C(═O), R¹²—C(═S), or R^(12′);

m¹ is 0 or 1;

R^(1′), R^(2′), and R^(3′) are each independently side chains of naturalor unnatural amino acids, alkyl, alkenyl, arylalkyl, aryl, alkoxy,alkoxycarbonyl, aryloxycarbonyl, alkylamino, or alkynyl, wherein atleast one of R^(2′) and R^(3′) comprise a carbamyl, hydroxyl orcarboxylate group; and

R¹² is C₁-C₄ alkyl,

R^(12′) is C₁-C₅ alkyl,

and pharmaceutically acceptable prodrugs and salts thereof, providedthat when D is acetyl, butanoyl or pentanoyl, then R^(3′) is not theside chain of Dab.

In yet another embodiment, the invention also includes polymyxinderivatives of formula (IV):

wherein:

A is a polymyxin B or polymyxin E ring moiety;

m¹ is 0 or 1;

L¹, L² and L³ are each independently C₁-C₃ alkyl or a covalent bond;

M¹, M² and M³ are each independently H, C(═O)NH₂, C(═O)OH, or —OH;

R¹² is C₁-C₄ alkyl,

and pharmaceutically acceptable prodrugs and salts thereof, providedthat when R¹² is methyl, propyl or butyl, then L³-M³ is not the sidechain of Dab, and wherein said derivative has three positive charges atphysiological pH.

In another embodiment, the invention also pertains to polymyxinderivatives of formula (V):

wherein R4 is an amino acid residue comprising a functional side chainable to cyclicize the molecule;

R6 and R7 are each independently selected optionally substitutedhydrophobic amino acid residues;

R10 is Leu or any non-hydrophobic amino acid residue; and

wherein R1 is optional; and wherein R1, R2, R3, R5, R8 and R9 are eachindependently selected amino acid residues; and wherein R(FA) is anoptionally substituted alkanoyl or alkyl residue having a total of 1 to5 carbon atoms; or a pharmaceutically acceptable salt or prodrugthereof, provided that (1) when R1 and R2 are absent, R3, R4. R5, R8,and R9 are Dab, R6 is D-Leu, R7 is L-Leu or L-Phe, and R10 is Thr, orwhen R1 and R2 are absent, R3, R4. R5, R8, and R9 are Dab, R6 is D-Phe,R7 is L-Leu, and R10 is Thr, then R(FA) is not an unsubstituted alkanoylresidue, and (2) when R(FA) is acetyl, butanoyl or pentanoyl, then R³ isnot Dab.

More specifically, the present invention relates to a derivative,wherein R2-R10 is selected from the group consisting ofThr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-] [=SEQ ID NO: 10 or 29] andThr-DAsn-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-] [=SEQ ID NO: 28].

The invention also relates to a combination product comprising two ormore of the derivatives according to the present invention, and to apharmaceutical composition comprising such derivative(s) or acombination thereof and pharmaceutically acceptable carriers andexcipients.

Furthermore, the present invention relates to a method for sensitizingGram-negative bacteria to an antibacterial agent, comprisingadministering, simultaneously or sequentially in any order, atherapeutically effective amount of said antibacterial agent and aderivative according to the present invention, wherein saidantibacterial agent may be selected from the group consisting ofclarithromycin, azithromycin, erythromycin and other macrolides,ketolides, clindamycin and other lincosamines, streptogramins, rifampin,rifabutin, rifalazile and other rifamycins, fusidic acid, mupirocin,oxazolidinones, vancomycin, dalbavancin, telavancin, oritavancin andother glycopeptide antibiotics, fluoroquinolones, bacitracin,tetracycline derivatives, betalactam antibiotics, novobiocin,pleuromutilins, folate synthesis inhibitors, deformylase inhibitors, andbacterial efflux pump inhibitors.

Also provided are methods for developing novel antibiotics; and forsensitizing clinically important Gram-negative bacteria to a hostdefense mechanism complement present in the serum.

The present invention also provides uses of a polymyxin derivativeaccording to the present invention in the manufacture of medicament forsensitizing Gram-negative bacteria, such e.g., Escherichia coli,Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae,Citrobacter freundii, and Acinetobacter baumannii against antibacterialagents; and for sensitizing Gram-negative bacteria to a host defensemechanism complement present in the serum.

The present invention also pertains to methods of treating Gram-negativeinfections in a subject comprising administering a derivative of theinvention (e.g., a derivative of formulae (I)-(V)) in combination withan antibacterial agent to a subject, such that the subject is treatedfor the infection.

Finally, the present invention relates to a process for preparing apolymyxin derivative according to the present invention, comprising (A)modifying a natural or synthetic polymyxin or octapeptin compound or aderivative thereof carrying 4 to 5 positively charged residues and aterminal moiety (D) comprising 1 to 5 carbon atoms by replacing 1 to 2of said positively charged residues by neutral residues or a covalentbond, or by converting 1 to 2 of said positively charged residues intoneutral residues in order to obtain a polymyxin derivative of formula(I) carrying 3 positively charged residues and a terminal moiety (D)comprising 1 to 5 carbon atoms, or (B) modifying a natural or syntheticpolymyxin or octapeptin compound or a derivative thereof carrying 4 to 5positively charged residues and a terminal moiety (D) comprising morethan 5 carbon atoms by replacing 1 to 2 of said positively chargedresidues by neutral residues or a covalent bond, or by converting 1 to 2of said positively charged residues into neutral residues, and byreplacing said terminal moiety (D) having more than 5 carbon atoms witha terminal moiety (D) comprising in total 1 to 5 carbon atoms in orderto obtain a polymyxin derivative of formula (1) carrying 3 positivelycharged residues and a terminal moiety (D) comprising in total 1 to 5carbon atoms, or (C) modifying a natural or synthetic polymyxin oroctapeptin compound or a derivative thereof carrying 4 to 6 positivelycharged residues and lacking the terminal moiety (D) by replacing 1 to 3of said residues by neutral residues, or by a covalent bond, orconverting 1 to 3 of said residues into neutral residues, and byintroducing a terminal moiety (D) comprising in total 1 to 5 carbonatoms, in order to obtain a polymyxin derivative of formula (I)according to claim 1, carrying 3 positively charged residues and anR(FA) having in total 1 to 5 carbon atoms. In one embodiment of theinvention, the terminal moiety D is R¹²—C(═O), R¹²—C(═S), or R^(12′),wherein R¹² and R^(12′) are defined hereinafter. In another embodiment,the terminal moiety (D) is R(FA), which is an optionally substitutedalkanoyl or alkyl residue having a total of 1 to 5 carbon atoms.

DEFINITIONS

“Physiological pH” as used herein refers to a pH value of more than 7.0and below 7.6, such as a pH value in the range of from 7.1 to 7.5, forexample in the range of from 7.2 to 7.4.

“Positive charge” as used herein denote positive charges at theabove-defined physiological pH.

“Cationic” molecule as used herein refers to a molecule that containsone or more positive charges.

“Amino acid residue” as used herein refers to any natural, non-naturalor modified amino acid residue, either in L- or D-configuration.

“Equivalent residues” as used herein, is intended to include obviousmodifications to e.g., amino acids, resulting in non-natural amino acidsor derivatives thereof, but retaining the structural and/or functionalcapacity of the replaced residue.

“Natural polymyxin(s)” as used herein, refers to polymyxins andcirculins.

“Polymyxin derivative” refers, for the purpose of this invention, tosynthetic or semisynthetic derivatives of natural polymyxins oroctapeptins, which have a cyclic heptapeptide (or heptapeptide ring)portion R4-R10 and a side chain linked to the N-terminal aminoacylresidue R4. The side chain may consist of an R(FA)-triaminoacyl(R1-R3),an R(FA)-diaminoacyl(R2-R3), an R(FA)-monoamino-acyl(R3), or of R(FA)alone.

“R(FA)” or “fatty acid tail” as used herein refers to the fatty acidpart, i.e., the alkanoyl part of the polymyxin structure, linked to theN-terminal amino acid residue of the linear peptide part (side chain) ofthe polymyxin or, in the absence of the linear peptide part, to theamino acid residue R4 (the amino acid in 4-position of the cyclicpeptide part of the polymyxin). Furthermore, for the purpose of thepresent invention, R(FA) may also be a related hydrophobic group, suchas alkyl. In certain embodiments of the invention, the fatty acid tailmay, in certain instances, be a terminal moiety selected from the groupconsisting of R¹²—(C═O); R¹²—SO₂—; R¹²—(C═NH)—; R¹²—NH—(C═S)—;R¹²—NH—(C═O)—; R¹²—NH—(C═NH)—;R¹²—O—(C═S)—; R¹²—O—(C═O); R¹²—P(O)OH—;R¹²—(C═S); and R^(12′), wherein R¹² and R^(12′) are alkyl, alkenyl,alkynyl, aryl, or aryl alkyl.

“Compounds” as used herein include all stereochemical isomers of saidcompound.

“Sensitizing activity” or “ability to sensitize” as used herein isintended to include any ability to increase the sensitivity, makesensitive or make susceptible a bacterium to an antibacterial agent.

“Polymyxin ring moiety” or “A” includes the ring portion of polymyxin A,polymyxin B, IL-polymyxin-B₁, polymyxin D, polymyxin E, polymyxin F,polymyxin M, polymyxin S, polymyxin T, circulin A, octapeptin A,octapeptin B, octapeptin C, octapeptin D, and derivatives thereof.Examples of derivatives include moieties with modifications which do notsubstantially effect the ability of the ring moiety to perform itsintended function, i.e., as an antibiotic and/or its ability tosensitize bacterium to one or more antibacterial agents. The term“polymyxin B ring moiety” refers to the ring portion of polymyxin B(i.e., cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-]). Other examples of polymyxinring moieties include moieties of the formula:

wherein:

R4 is an amino acid residue comprising a functional side chain able tocyclicize the molecule;

R5, R8, and R9 are independently selected amino acid residues;

R6 and R7 are optionally substituted hydrophobic amino acid residues;and

R10 is Leu or any non-hydrophobic amino acid residue. Other examples ofR4-R10 are discussed in further detail in Formula (V).

The term “octapeptin ring” refers to the ring portion of nativeoctapeptin A (i.e., cy[Dab-Dab-DLeu-LLeu-Dab-Dab-Thr-], i.e., compoundswherein R4, R5, R8, and R9 are Dab, R6 is DLeu, R7 is LLeu, and R10 isThr), octapeptin B (i.e., cy[Dab-Dab-DLeu-LPhe-Dab-Dab-Thr-], i.e.,compounds wherein R4, R5, R8 and R9 are Dab, R6 is DLeu, R7 is LPhe andR10 is Thr), and octapeptin C (i.e., cy[Dab-Dab-DPhe-LLeu-Dab-Dab-Thr-],i.e., compounds wherein R4, R5, R8 and R9 are Dab, R6 is DPhe, R7 isLLeu, and R10 is Thr).

The term “prodrug” includes moieties which are cleaved in vivo to yieldan active polymyxin derivative compound of the invention. The prodrugsinclude moieties which mask or otherwise neutralize the positive charges(i.e., the —NH₃ ⁺ or other protonated species) at physiological pH. Oncethe prodrug is administered to the subject, the prodrug moieties orcharge masking moieties will be cleaved or other wise removed to yieldthe active polymyxin derivative of the invention, optionally with threepositive charges at physiological pH.

The term “charge masking moiety” includes moieties that reversiblyneutralize positive charges on the derivatives. Preferably, the moietiesare cleaved or otherwise disassociated with the positive charges of thepolymyxin compound after being administered to a subject. Examples ofcharge masking moieties include sulfoalkyl (e.g., sulfomethylatedderivatives). Other positive charge masking moieties include, but arenot limited to, chloride, bromide, iodide, nitrate, sulfate, bisulfate,phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate,citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)).

The term “subject” includes organisms capable of suffering frombacterial infections. Examples of subjects include mammals, e.g.,horses, cows, pigs, sheep, goats, cats, dogs, rabbits, ferrets, monkeys,and, preferably, humans.

ABBREVIATIONS

Fatty acids: FA, fatty acyl residue; 6-MOA and MOA, 6-methyloctanoylresidue; 6-MHA and MHA, 6-methylheptanoyl residue; MO(H)A, the mixtureof 6-methyloctanoyl, 6-methylheptanoyl and related fatty acyl residuesoccurring in polymyxin B; OHMDA, 3-OH-8-methyldecanoic acid; OA,octanoyl; DA, decanoyl; Ac, acetyl; Me, methyl.

Amino acids: Dab, α,γ-diamino-n-butyryl residue; fDab, N-γ-formyldiamino-n-butyryl residue; acDab, N-γ-acetyldiamino-n-butyryl residue;Abu, α-aminobutyryl residue; Asn, aspartyl residue; Thr, threonylresidue; Ser, serinyl residue; Phe, phenylalanyl residue; Leu, leucylresidue; Ile, isoleucyl residue; Ala, alanyl residue; sm-Dab,γ-sulphomethylated α,γ-diamino-n-butyryl residue. One-letter codes formodified amino acyl residues: X, Dab; Z, Abu; B, N-γ-fDab; J, N-γ-acDab.

Peptides: DAPB, deacylpolymyxin B; DAC, deacylcolistin; PMBN, polymyxinB nonapeptide; PMEN, polymyxin E nonapeptide; PMBO, polymyxin

B octapeptide; PMHP, polymyxin B heptapeptide.

Other: cy, cyclo (to denote the cyclic part of the peptide, enclosedwithin brackets); f, formyl; ac, acetyl; sm, sulfomethyl; MS,methanesulfonate; LPS, lipopolysaccharide; OM, outer membrane; MIC,minimum inhibitory concentration; CFU, colony forming unit. The symbol *is used herein to mark the residues between which the heptapeptide ringportion of the compound is closed leaving the remaining part of themolecule as a side chain.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that certain polymyxin-like compounds containingonly three (3) positive charges and having only a short fatty acyl tailR(FA) or terminal moiety (D) (not more than 5 carbon atoms in total)still possess the ability to sensitize Gram-negative bacteria toantibacterial agents such as antibiotics, semisynthetic antibiotics andchemotherapeutic agents as well as to host defence factors such as thecomplement system of fresh human serum.

Because these novel compounds do not have more than three (3) positivecharges, they, in analogy with the polymyxin derivatives described inU.S. patent application Ser. No. 11/891,629, may be less toxic ingeneral and less nephrotoxic in particular than polymyxins and theirknown derivatives. Similarly, the compounds now invented may reduce lesshistamine from the host tissues than and have pharmacokinetic propertiesadvantageous over polymyxin B, colistin, and their previously describedderivatives. Furthermore, the short R(FA) or terminal moiety (D) maymake the novel compounds less toxic in acute toxicity assays, in analogywith polymyxin B nonapeptide and colistin nonapeptide that lack theentire fatty acyl part. Furthermore, the novel compounds may havepharmacokinetic properties that are advantageous over polymyxinderivatives that have a long R(FA) or a terminal moiety (D) with morethan five carbon atoms.

In one embodiment, the invention pertains to polymyxin derivatives ofthe formula (I):

wherein:

A is a polymyxin ring moiety;

D is a terminal moiety comprising 1 to 5 carbon atoms;

m¹, m², and m³ are each independently 0 or 1;

Q¹, Q², and Q³ are each independently CH₂, C═O, or C═S;

W¹, W², and W³ are each independently NR⁴, O, or S;

R^(1′), R^(2′), and R^(3′) are each independently side chains of naturalor unnatural amino acids, alkyl, alkenyl, arylalkyl, aryl, alkoxy,alkoxycarbonyl, aryloxycarbonyl, alkylamino, or alkynyl; and

R⁴ is hydrogen or alkyl,

and pharmaceutically acceptable prodrugs and salts thereof, providedthat (1) when A is an octapeptin ring, m¹ and m² are 0, m³ is 1, W³ isNH, Q³ is C═O, and R^(3′) is the side chain of diaminobutyric acid(Dab), then D is not C₂-C₅ acyl, and (2) when D is acetyl, butanoyl orpentanoyl, then R^(3′) is not the side chain of Dab.

In certain embodiments, the compounds of the invention (e.g.,derivatives of any one of formulae (I)-(V)) may have at least two but nomore than three positive charges at physiological pH. In anotherembodiment, the compounds have three positive charges at physiologicalpH.

Examples of prodrugs of these derivatives include those with chargemasking moieties which neutralize the three positive charges whenadministered to the subject which are removed in vivo to yield thecompound with three positive charges. Examples of charge maskingmoieties include sulfoalkyl moieties such as sulfomethyl.

Preferably, the derivatives have three positive charges at physiologicalpH, as defined above. In certain embodiments of the invention, R^(1′),R^(2′), and R^(3′) do not comprise positively charged functional groupsat physiological pH. R^(1′), R^(2′), and R^(3′) may comprise, forexample, one or two or more hydroxyl, carboxylate, carbamyl, thiol,sulfate, sulfonyl, or phosphate groups.

In one embodiment, m¹ is 0 and m² and m³ are each 1. In another, Q² andQ³ are each C═O and W² and W³ are each NH.

In certain embodiments, R^(2′) is substituted with one or more groupsselected from hydroxyl, carbamyl, carboxylate, thiol, sulfate, sulfonyl,or phosphate groups. Preferably, R^(2′) is substituted with a carbamyl,hydroxyl or carboxylate group. Examples of R^(2′) include substitutedalkyl and the side chains of alanine, aminobutyric acid, asparagine,aspartic acid, diaminobutyric acid, glutamic acid, glutamine, serine, orthreonine in either the D- or L-configuration. Preferably, R^(2′) isD-alanine, L-serine, or L-threonine.

In certain embodiments, R^(3′) is substituted with one or more groupsselected from carbamyl, hydroxyl, carboxylate, thiol, sulfate, sulfonyl,or phosphate. Preferably, R^(3′) is substituted alkyl and maybesubstituted with a carbamyl, hydroxyl or carboxylate group. R^(3′) maybe the side chain of alanine, aminobutyric acid, asparagine, asparticacid, diaminobutyric acid, glutamic acid, glutamine, serine, orthreonine in either the D- or L-configuration. Preferably, R^(3′) isD-asparagine, L- or D-serine.

Examples of A include the ring moiety of polymyxin B (i.e.,cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-]) and polymyxin E (i.e.,cy[Dab-Dab-DLeu-Leu-Dab-Dab-Thr-].

In a further embodiment, the terminal moiety is selected from the groupconsisting of R¹²—(C═O); R¹²—SO₂—; R¹²—(C═NH)—; R¹²—NH—(C═S)—;R¹²—NH—(C═O)—; R¹²—NH—(C═NH)—;R¹²—O—(C═S)—; R¹²—O—(C═O); R¹²—P(O)OH—;R¹²—(C═S); or R^(12′), wherein R¹² and R^(12′) are each alkyl,cycloalkyl, alkenyl, alkynyl, aryl, or aryl alkyl. In certainembodiments, D is R¹²—(C═O) or R¹²—(C═S) and R¹² is methyl, ethyl,propyl, or butyl. Specific examples of D include acetyl, propionyl,butanoyl, and pentanoyl.

In another embodiment, the invention also pertains to polymyxinderivatives of formula (II):

wherein:

A is a polymyxin ring moiety;

D is R¹²—C(═O), R¹²—C(═S) or R^(12′);

m¹, m², and m³ are each independently 0 or 1, provided that at least oneof m¹, m², and m³ are 1;

R^(1′), R^(2′), and R^(3′) are each independently side chains of naturalor unnatural amino acids, alkyl, alkenyl, arylalkyl, aryl, alkoxy,alkoxycarbonyl, aryloxycarbonyl, alkylamino, or alkynyl; and

R¹² is C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl,

R^(12′) is C₁-C₅ alkyl, C₂-C₅ alkenyl, or C₂-C₅ alkynyl,

and pharmaceutically acceptable prodrugs and salts thereof, providedthat (1) when A is an octapeptin ring, m¹ and m² are 0, m³ is 1, andR^(3′) is the side chain of diaminobutyric acid (Dab), and D is R¹²—C═O,then R¹² is not C₁-C₅ alkyl, and (2) when D is acetyl, butanoyl orpentanoyl, then R^(3′) is not the side chain of Dab.

Preferably, the derivative of formula (II) has three positive charges atphysiological pH. In a further embodiment, m¹ may be 0 and/or m² and m³may each be 1. In a further embodiment, R^(2′) and/or R^(3′) may eachindependently be substituted alkyl (e.g., substituted with a carbamyl,hydroxyl or carboxylate group). Furthermore, R^(2′) and/or R^(3′) mayeach be the side chain of serine or threonine (including both D and Lconfigurations). Examples of R^(2′) include the side chains ofD-alanine, L-serine and L-threonine. Examples of R^(3′) include the sidechains of D-asparagine, L- and D-serine.

In a further embodiment, R¹² is alkyl and D may be acetyl, propionyl,butanoyl, or pentanoyl.

In another further embodiment, the invention also pertains to polymyxinderivatives of formula (III):

wherein:

A is a polymyxin B or polymyxin E ring moiety;

D is R¹²—C(═O), R¹²—C(═S) or R^(12′);

m¹ is 0 or 1;

R^(1′), R^(2′), and R^(3′) are each independently side chains of naturalor unnatural amino acids, alkyl, alkenyl, arylalkyl, aryl, alkoxy,alkoxycarbonyl, aryloxycarbonyl, alkylamino, or alkynyl, wherein atleast one of R^(2′) and R^(3′) comprise a carbamyl, hydroxyl orcarboxylate group; and

R¹² is C₁-C₄ alkyl,

R^(12′) is C₁-C₅ alkyl,

and pharmaceutically acceptable prodrugs and salts thereof, providedthat when D is acetyl, butanoyl or pentanoyl, then R^(3′) is not theside chain of Dab.

Preferably, the compounds of the invention have three positive chargesat physiological pH, m¹ is 0, R^(2′) and R^(3′) are both substitutedalkyl, and/or D is acetyl, propionyl, butanoyl, or pentanoyl.

In yet another embodiment, the invention also features a polymyxinderivative of formula (IV):

wherein:

A is a polymyxin B or polymyxin E ring moiety;

m¹ is 0 or 1;

L¹, L² and L³ are each independently C₁-C₃ alkyl or a covalent bond;

M¹, M² and M³ are each independently H, C(═O)NH₂, C(═O)OH, or —OH;

R¹² is C₁ -C₄ alkyl,

and pharmaceutically acceptable prodrugs and salts thereof, providedthat when R¹² is methyl, propyl or butyl, then L³-M³ is not the sidechain of Dab.

Preferably, m¹ is 0. Examples of L² include branched alkyl (e.g.,—CH(CH₃)—). Examples of M² include OH. Other examples of L² include—CH₂— and other examples of M² include OH and H. In another embodiment,L³ is —CH₂— and M³ is OH. In yet another embodiment, L³ is —CH₂—CH₂— andM³ is C(═O)NH₂. Preferably, the compounds of formula (IV) have threepositive charges at physiological pH.

The present invention thus relates to a polymyxin derivative which maybe represented by the general formula (V):

wherein

R4 is an amino acid residue comprising a functional side chain able tocyclicize the molecule;

R6 and R7 are an optionally substituted hydrophobic amino acid residues;

R10 is Leu or any non-hydrophobic amino acid residue; and

wherein R1 may be absent; and

wherein R1, R2, R3, R5, R8 and R9 are each independently selected aminoacids; and

wherein R(FA) is an optionally substituted alkanoyl or alkyl residue,having in total 1 to 5 carbon atoms.

or a pharmaceutically acceptable prodrug or salt thereof provided that(1) when R1 and R2 are absent, R3, R4. R5, R8, and R9 are Dab, R6 isD-Leu, R7 is L-Leu or L-Phe, and R10 is Thr or when R1 and R2 areabsent, R3, R4. R5, R8, and R9 are Dab, R6 is D-Phe, R7 is L-Leu, andR10 is Thr, then R(FA) is not an unsubstituted alkanoyl residue and (2)when R(FA) is acetyl, butanoyl or pentanoyl, then R³ is not Dab.

In a derivative according to the present invention, R(FA) may be anyresidue that has small molecular weight and 1 to 5 carbon atoms. Themajor role of a short R(FA) is to block the free N-terminal amino groupof the peptide and thus eliminate one positive charge of the peptide.

Preferably, the compounds of formula (V) may have three positive chargesat physiological pH. Furthermore, R1, R2, R3, R5, R8 and R9 may bespecifically selected such that the compounds have three positivecharges at physiological pH.

The R(FA) is preferably selected from the group consisting of carboxylicacid residues, i.e., alkanoyl groups, or alkyl groups, having in total 1to 5 carbon atoms. R(FA) is preferably selected from the groupconsisting of methyl, formyl and acetyl residues. Other useful residuesmay be selected from propanoyl, butanoyl, isobutanoyl, valeroyl, andisovaleroyl residues. The residues may be branched, straight-chained orcyclic.

R(FA) may also be an unsaturated residue, containing one or more doubleor triple bonds.

R(FA) may be substituted with substituents readily recognizable by oneskilled in the art, provided that R(FA) has no more than 1 to 5 carbonatoms. The substituents may include alkyl, hydroxy and alkoxy. Alkyl ispreferably methyl, ethyl, or propyl. Alkoxy is preferably methoxy,ethoxy, or propoxy. A person skilled in the art may readily recognizeequivalents of these preferred R(FA) residues and substituents thereof.

In natural polymyxins and octapeptins, R1 is Dab or absent (i.e.,replaced by a covalent bond). Examples of known derivatives that haveantibacterial activity include those wherein R1 is Ala or a covalentbond.

In a derivative according to the present invention R1, if present, maybe any amino acid residue, provided that the total number of positivecharges in said derivative does not exceed three and that the totalnumber of positive charges in the side chain portion does not exceedone, and is preferably absent.

In natural polymyxins and octapeptins, R2 is Thr or absent (i.e.,replaced by a covalent bond). Examples of known derivatives that haveantibacterial activity include those wherein R2 is O-acetyl-Thr,O-propionyl-Thr, O-butyryl-Thr or a covalent bond.

In a derivative according to the present invention, R2 may be any aminoacid residue, preferably hydrophilic or relative hydrophilic, providedthat the total number of positive charges in said derivative does notexceed three and that the total number of positive charges in the sidechain portion does not exceed one. Examples of R2 include alanine,aminobutyric acid, asparagine, aspartic acid, diaminobutyric acid,glutamic acid, glutamine, serine, or threonine in either D- or L-configuration, A person skilled in the art may also recognize anequivalent residue of Thr to be Ser.

In natural polymyxins and octapeptins, R3 is Dab, DDab or DSer.

In a derivative according to the present invention, R3 may be any aminoacid residue, preferably hydrophilic or relatively hydrophilic, providedthat the total number of positive charges in said derivative does notexceed three and that the total number of positive charges in the chainportion does not exceed one, and is selected from the group consistingof alanine, amino-butyric acid, asparagine, aspartic acid,diaminobutyric acid, glutamic acid, glutamine, serine, or threonine ineither D- or L-configuration.

A person skilled in the art may readily recognize other hydrophilic orrelatively hydrophilic residues than these preferred residues R1, R2 andR3, and may select such from a group consisting of e.g. arginine,N_(ω)-methyl arginine, α-methylaspartate, cysteine, histidine,hydroxylysine, lysine, methionine, ornithine, penicilamine, proline,phosphoserine, phosphothreonine, and tyrosine.

A person skilled in the art may readily note that one of the residuesR1, R2 and R3 may not be hydrophilic or relatively hydrophilic, providedthat the other two residues are so. Accordingly, R1, R2 and R3 may beselected from a group consisting of e.g. a covalent bond, alanine,2-aminoadipic acid, α-n-butyric acid, N-(4-aminobutyl)glycine,α-aminobutyric acid, γ-aminobutyric acid, α-amino-caproic acid,aminocyclopropanecarboxylate, aminoisobutyric acid,aminonorbornylcarboxylate, α-amino-n-valeric acid, arginine,N_(ω)-methyl arginine, asparagine, α-methylaspartate, aspartic acid,N-benzylglycine, N-(2-carbamylethyl)glycine, N-(carbamylethyl)glycine,1-carboxy-1(2,2-diphenyl ethylamino)cyclopropane, cysteine,N_(α)-methyldiamino-n-butyric acid, N_(γ)-acetyldiamino-n-butyric acid,N_(γ)-formyldiamino-n-butyric acid, N_(γ)-methyldiamino-n-butyric acid,N—(N-2,2-diphenylethyl)carbamylmethyl-glycine,N—(N-3,3-diphenylpropyl)carbamylmethyl(1)glycine, N-(3,3-diphenylpropyl)glycine, glutamic acid, glutamine, glycine, t-butylglycine,2-amino-4-guanidinobutyric acid, N-(3-guanidinopropyl)glycine,histidine, homophenylalanine, isodesmosine, isoleucine, leucine,norleucine, hydroxylysine, N_(α)-methyllysine, lysine,N_(α)-methylhydroxylysine, N_(α)-methyllysine,N_(ε)-acetylhydroxylysine, N_(ε)-acetyl lysine,N_(ε)-formylhydoxylysine, N_(ε)-formyllysine, N_(ε)-methylhydroxylysine,N_(ε)-methyllysine, methionine, α-methyl-γ-aminobutyrate,α-methyl-aminoisobutyrate, α-methylcyclohexylalanine, α-napthylalanine,norleucine, norvaline, α-methylornithine, N_(α)-methylornithine,N_(δ)-acetylornithine, N_(δ)-formyl-ornithine, N_(δ)-methylornithine,ornithine, penicilamine, phenylalanine, hydroxyproline, proline,N_(α)-methyldi-amino-n-propionic acid, N_(β)-acetyldiamino-n-propionicacid, N_(β)-formyldiamino-n-propionic acid,N_(β)-methyldiamino-n-propionic acid, phosphoserine, serine,phosphothreonine, threonine, tryptophan, tyrosine, norvaline, andvaline.

In natural polymyxins and octapeptins, R4 is Dab. Examples of syntheticderivatives that have antibacterial activity include those wherein R4 isLys.

In a derivative according to the present invention R4 is an amino acidresidue comprising a functional side chain able to cyclicize themolecule, and may be selected from the group of equivalent residuesconsisting of Lys, hydroxylysine, ornithine, Glu, Asp, Dab,diaminopropionic acid, Thr, Ser and Cys, preferably Dab.

In natural polymyxins and octapeptins, R5, R8 and R9 are Dab. Examplesof synthetic derivatives that have antibacterial activity include thosewherein R5, R8, and R9 may be Lys or 2-amino-4-guanidino butyric acid.

In a derivative according to the present invention R5, R8 and R9 may bea positively charged or a neutral amino acid residue, preferably Dab,provided that the total number of positive charges in said derivativedoes not exceed three.

A person skilled in the art, may readily recognize equivalent residuesof these preferred residues, and may select such from a group consistingof e.g. diaminobutyric acid, diaminopropionic acid, lysine,hydroxylysine, ornithine, 2-amino-4-guanidinobutyric acid, glycine,alanine, valine, leucine, isoleucine, phenylalanine, D-phenylalanine,methionine, threonine, serine, α-amino-n-butyric acid, α-amino-n-valericacid, α-amino-caproic acid, N_(ε)-formyllysine, N_(ε)-acetyllysine,N_(ε)-methyllysine, N_(ε)-formylhydroxylysine, N_(ε)-acetylhydroxylysine, N_(ε)-methylhydroxylysine, L-N_(α)-methylhydroxylysine,N_(γ)-formyl diamino-n-butyric acid, N_(γ)-acetyldiamino-n-butyric acid,N_(γ)-methyldiamino-n-butyric acid, N_(β)-formyldiamino-n-propionicacid, D-N_(β)-formyldiamino-n-propionic acid,N_(β)-acetyldiamino-n-propionic acid, N_(β)-methyldiamino-n-propionicacid, N_(δ)-formylornithine, N_(δ)-acetylornithine andN_(δ)-methylornithine.

In natural polymyxins and octapeptins, R6 is DPhe or DLeu and R7 is Leu,Ile, Phe or Thr. Synthetic derivatives that have antibacterial activityinclude those wherein R6 is DTrp and wherein R7 is Ala.

In a derivative according to the present invention, R6 is an optionallysubstituted hydrophobic amino acid residue, preferably DPhe or DLeu, andR7 is an optionally substituted hydrophobic residue, preferably Leu, Thror Ile.

A person skilled in the art may readily recognize equivalent residues ofthese preferred hydrophobic residues, and may select such from a groupconsisting of e.g. phenylalanine, α-amino-n-butyric acid, tryptophane,leucine, methionine, valine, norvaline, norleucine, isoleucine andtyrosine. A person skilled in the art may also recognize the equivalentresidue of threonine to be serine.

In natural polymyxins and octapeptins, R10 is Thr and Leu. Examples ofknown derivatives that have antibacterial activity include those whereinR10 is O-acetyl-Thr, O-propionyl-Thr or O-butyryl-Thr.

In a derivative according to the present invention, R10 is Leu or anynon-hydrophobic amino acid residue, provided that that the total numberof positive charges in said derivative does not exceed three. PreferablyR10 is Thr or Leu.

A person skilled in the art may also recognize the equivalent residue ofthreonine to be serine.

The three (3) positive charges present in the derivatives according tothe invention can be located in the heptapeptide ring portion; or two(2) positive charges can be located in heptapeptide ring portion whilethe remaining one positive charge is located in the side chain.

In one embodiment, derivatives according to the present invention can beselected from the group of derivatives wherein R2-R10 is selected fromthe group consisting of Thr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-],i.e., SEQ ID NO: 10 or 29; andThr-DAsn-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e., SEQ ID NO: 28.

In other embodiments, derivatives according to the present invention canbe selected from the group consisting of:acetyl-Thr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e., Ac-SEQ ID NO:10; and acetyl-Thr-DAsn-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e., Ac-SEQID NO: 28.

As shown in the example section herein, the compounds according to thepresent invention carrying only three (3) positive charges and having anR(FA) containing 1 to 5 carbon atoms only can be very potent agents tosensitize Gram-negative bacteria to antibacterial agents.

For sensitizing activity at least two (2) and more preferably three (3)positive charges are located in the heptapeptide ring part.

The works of Teuber (1970), Srinivasa and Ramachandran (1980a), andSakura et al. (2004) disclose, among other polymyxin derivatives,derivatives having only two (2) or three (3) positive charges. However,the compounds carry a fatty acid part R(FA) longer than 5 carbon atoms.On the other hand, polymyxin B nonapeptide and colistin nonapeptide,both previously known effective agents to sensitize Gram-negativebacteria to antibiotics, lack the entire R(FA) part but carry five (5)positive charges.

In certain embodiments of the invention, the polymyxin derivatives offormulae I-V may be administered to a subject in prodrug form. Theprodrug may comprise one or more charge masking moieties which mask thepositive charges of the compound until after it is administered to thesubject.

The present invention in one aspect provides new polymyxin derivativescarrying three (3) positive charges only and an R(FA) containing 1 to 5carbon atoms only and being capable of sensitizing one or moreGram-negative bacterial species to an antibiotic or antibacterial agent.

The susceptibility of bacteria to an antibacterial agent may bedetermined by two microbiological methods. A rapid but crude procedureuses commercially available filter paper disks that have beenimpregnated with a specific quantity of the antibacterial agent. Thesedisks are placed on the surface of agar plates that have been inoculatedwith a suspension of the organism being tested, and the plates areobserved for zones of growth inhibition. A more accurate technique, thebroth dilution susceptibility test, involves preparing test tubescontaining serial dilutions of the drug in liquid culture media, theninoculating the organism being tested into the tubes. The lowestconcentration of drug that inhibits growth of the bacteria after asuitable period of incubation is reported as the minimum inhibitoryconcentration (MIC).

Derivatives according to the present invention may sensitize clinicallyimportant Gram-negative bacteria to antibacterial agents, where saidGram-negative bacteria may be those belonging to the genus ofAcinetobacter, Aeromonas, Alcaligenes, Bordetella, Branhamella,Campylobacter, Citrobacter, Enterobacter, Escherichia, Francisella,Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella,Moraxella, Pasteurella, Plesiomonas, Pseudomonas, Salmonella, Serratia,Shigella, and Yersinia species. The bacteria may be, for example,herichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobactercloacae, Enterobacter aerogenes, other species of Enterobacter,Citrobacter freundii, Acinetobacter baumannii, Pseudomonas aeruginosaand other Pseudomonas species as well as many other species ofnon-fermentative Gram-negative bacteria. The bacteria also includeHelicobacter pylori, as well as other clinically important Gram-negativebacteria.

The bacterial infections to be treated include, for example, bacteremia,septicemia, skin and soft tissue infection, pneumonia, meningitis,infections in the pelveoperitoneal region, foreign body infection, feverin hematological patient, infection associated with an intravenous lineor other catheter, canyl and/or device, infection in gastrointestinaltract, in the eye, or in the ear, superficial skin infection, andcolonization of gastrointestinal tract, mucous membranes and/or skin bypotentially noxious bacteria.

The bacterial infectious diseases include (but are not limited to)severe hospital-acquired infections, infections of the immunocompromisedpatients, infections of the organ transplant patients, infections at theintensive care units (ICU), severe infections of burn wounds, severecommunity-acquired infections, infections of cystic fibrosis patients,as well as infections caused by multi-resistant Gram-negative bacteria.

The present invention is also directed to combinations of two or morederivatives according to the present invention for combinationtreatment. The combinations may include derivatives having a capabilityto sensitize different species or strains of Gram-negative bacteria toantibacterial agents.

Another aspect of the present invention is directed to pharmaceuticalcompositions comprising polymyxin derivatives according to the presentinvention, their prodrug and salt forms, selected combinations thereof,and optionally an antibacterial agent formulated together with one ormore pharmaceutically acceptable carriers and excipients. Theyfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically and include e.g. diluting, filling,buffering, thickening, wetting, dispersing, solubilizing, suspending,emulsifying, binding, stabilizing, disintegrating, encapsulating,coating, embedding, lubricating, colouring, and flavouring agents aswell as absorbents, absorption enhancers, humefactants, preservativesand the like, well-known to a person skilled in the art.

Pharmaceutical compositions include compositions wherein the activeingredients are contained in an amount effective to achieve the intendedpurpose. More specifically, a therapeutically effective amount inconnection with the present invention means an amount of compoundeffective to sensitize Gram-negative bacteria to antibacterial agents.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art of medicine.

Compositions may be produced by processes well known in the art, e.g. bymeans of conventional mixing, dissolving, encapsulating, entrapping,lyophilizing, emulsifying and granulating processes. The properformulation is dependent upon the route of administration chosen, andthe pharmaceutical composition can be formulated for immediate releaseor slow release (e.g. in order to prolong the therapeutic effect and/orimprove tolerability). Furthermore, the formulations may conveniently bepresented in unit dosage form by methods known in the art of pharmacy.

Pharmaceutical compositions according to the present invention include(but are not limited to) those intended for intravenous, intramuscular,oral, or topical administration as well as those being administered as asuppositorium or as an inhalable aerosol. The compositions includeintravenous, intramuscular, intraperitoneal, subcutaneous,intramedullary, intrathecal, intraventricular, intranasal, orintraocular injections, inhalable aerosols as well as those intended forrectal, oral, intravaginal, transmucosal or transdermal delivery.

For parenteral administration (e.g. by bolus injection, fast runninginfusions, or slow infusions), the compounds according to this inventionas well as the combinations described above may be formulated as theirsuitable salt or ester forms in sterile aqueous solutions, preferablyphysiologically compatible fluids such as saline, 5% dextrose, Ringer'ssolution, and Hank's solution. The formulation may also include organicsolvents such as propylene glycol, polyethylene glycol, propylene glycolor related compounds as well as preservatives and surfactants.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

In addition, the pharmaceutical compositions for parental administrationmay be suspensions or emulsions in oily or aqueous vehicles, and maycontain formulatory agents such as suspending, stabilizing and/ordispersing agents. Suitable lipophilic vehicles and solvents includefatty oils such as natural and/or synthetic fatty acids esters, such asethyl oleate and triglycerides, or liposomes. The suspensions maycontain substances, which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol or dextran.

The parenteral compositions can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use.

For oral administration, solid form preparations include e.g. powders,tablets, pills, dragees, lozenges, capsules, cachets, and microgranularpreparations. Pharmaceutical preparations can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. A solid carrier/excipient can be one ormore substances which may also act as diluents, solubilizers,lubricants, suspending agents, binders, preservatives, flavouringagents, wetting agents, tablet disintegrating agents, or anencapsulating material. Suitable carriers include, but are not limitedto, magnesium carbonate, magnesium stearate, talc, dextrose, lactose,pectin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.

Liquid preparations suitable for oral administration include, e.g.,aqueous solutions, syrups, elixirs, aqueous suspensions, emulsions andgels. Aqueous solutions can be prepared by dissolving the activecomponent in water and adding suitable stabilizing and thickening agentsas well as colorants and flavours. Aqueous suspensions can be preparedby dispersing the finely divided active component in water with viscousmaterial, such as natural or synthetic gums, resins, methylcellulose,sodium carboxymethylcellulose, and other well known suspending agents.Emulsions may be prepared in solutions in aqueous propylene glycolsolutions or may contain emulsifying agents such as lecithin, sorbitanmonooleate or acacia.

The compounds according to the invention or combinations described abovemay also be formulated for topical administration. The active compoundsare admixed under sterile conditions with pharmaceutically acceptablecarriers/excipients, including any needed buffering agents andpreservatives. Ointments, creams and lotions may, for example, beformulated with an aqueous or oily base with the addition of suitableemulsifying, dispersing, suspending, thickening, stabilizing, orcoloring agents. Commonly used excipients include animal and vegetablefats and oils, waxes, paraffins, starch, cellulose derivatives,tragacanth, and polyethylene glycol.

Other topical formulations include, but are not limited to, ear-drops,eye-drops and transdermal patches.

For transdermal as well as transmucosal administration, penetrantsgenerally known in the art may be used in the formulation.

For administration by inhalation, the compounds according to thisinvention and the combinations described above are delivered in the formof an aerosol spray presentation from a ventilator, pressurized pack ora nebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds according to this invention and the combinations describedabove may also be formulated in rectal compositions such as retentionenemas or suppositories, using conventional suppository bases such ascocoa butter, other glycerides, polyethylene glycol, or a suppositorywax.

The present invention also relates to a method for using the presentpolymyxin derivatives or a combination of such derivatives as a part ofthe clinical treatment of (or a preventive prophylactic regimen for)human or animal subjects suffering of an infectious disease (i.e., aGram-negative bacterial infection), and comprises administering to saidsubject an therapeutically effective dose of at least one derivativeaccording to the present invention, in combination with an antibacterialagent.

The present invention also relates to a method of sensitizingGram-negative bacteria to an antibacterial agent, wherein the derivativeaccording to the present invention is administered simultaneously, orsequentially in any order, with a therapeutically effective amount ofsaid antibacterial agent.

The derivative of the present invention and the antibacterial agent maybe administered together as one formulation or by different routes. Forexample, the polymyxin derivative may be administered intravenouslywhile the antibacterial agent is administered intramuscularly,intravenously, subcutaneously, orally or intraperitoneally.Alternatively, the derivative may be administered intramuscularly orintraperitoneally while the antibacterial agent is administeredintravenously, intramuscularly or intraperitoneally, or the derivativemay be administered in an aerosolized or nebulized form while theantibacterial agent is administered, e.g., intravenously. The derivativeand the antibacterial agents may be administered simultaneously orsequentially, as long as they are given in a manner sufficient to allowboth to achieve effective concentrations at the site of infection.

“Therapeutic effectiveness” is based on a successful clinical outcome,and does not require that a derivative according to the presentinvention, in combination with an antibacterial agent, kills 100% of thebacteria involved in an infection. Successful treatment depends onachieving a level of antibacterial activity at the site of infection,sufficient to inhibit the bacteria in a manner that tips the balance infavor of the host. When host defenses are maximally effective, theantibacterial effect required may be modest. Reducing organism load byeven one log (a factor of 10) may permit the host's own defenses tocontrol the infection. In addition, augmenting an earlybactericidal/bacteriostatic effect can be more important than long-termbactericidal/bacteriostatic effect. These early events are a significantand critical part of therapeutic success, because they allow time forhost defense mechanisms to activate. Increasing the bactericidal ratemay be particularly important for infections such as meningitis, bone orjoint infections.

The therapeutic effectiveness of an antibacterial agent depends on thesusceptibility of the bacterial species to said antibacterial agent atthe clinically relevant concentration of the derivative according tothis invention. The effect of compounds according to the presentinvention to improve the therapeutic effectiveness of antibacterialagents in vivo may be demonstrated in vivo animal models, such as mouseperitonitis or rabbit bacteremia assays, and may be predicted on thebasis of a variety of in vitro tests, including (1) determinations ofthe minimum inhibitory concentration (MIC) of an antibacterial agentrequired to inhibit growth of a Gram-negative bacterium for 24 hours,(2) determinations of the effect of an antibacterial agent on thekinetic growth curve of a Gram-negative bacterium, and (3) checkerboardassays of the MIC of serial dilutions of antibacterial agent alone or incombination with serial dilutions of compound(s). Exemplary models ortests are well known in the art.

Using in vitro determinations of MIC at 24 hours, a derivative accordingto the present invention may be shown to reduce the MIC of theanti-bacterial agent. With this result, it is expected that concurrentadministration of the compound in vivo will increase susceptibility of aGram-negative bacterium to the antibacterial agent. A compound accordingto the present invention may also be shown to reduce the MIC of anantibacterial agent from the range in which the organism is consideredclinically resistant to a range in which the organism is consideredclinically susceptible. With this result, it is expected that concurrentadministration in vivo of the one or more compound(s) according to thepresent invention with the antibacterial agent will reverse resistanceand effectively convert the antibiotic-resistant organism into anantibiotic-susceptible organism.

By measuring the effect of antibacterial agents on the in vitro growthcurves of Gram-negative bacteria, in the presence or absence of acompound according to the present invention, the compound may be shownto enhance the early antibacterial effect of antibacterial agents withina period of preferably less than 24 hours. Enhancement of earlybactericidal/growth inhibitory effects is important in determiningtherapeutic outcome.

In a checkerboard assay, the combination of a compound according to thepresent invention with antibacterial agents may result in a“synergistic” fractional inhibitory concentration index (FICI). Thecheckerboard method is based on additivity, which assumes that theresult observed with multiple drugs is the sum of the separate effectsof the drugs being tested; according to this system a FICI of less than0.5 is scored as synergy, 1 is scored as additive, and greater than 1but less than 2 is scored as indifferent.

Antibacterial agents suitable for use in combination with derivativesaccording to the present invention, include e.g. macrolides, such asclarithromycin, azithromycin, and erythromycin, ketolides, lincosamines,such as clindamycin, streptogramins, rifamycins, such as rifampin,rifabutin and rifalazile, fusidic acid, mupirocin, oxazolidinones,glycopeptide antibiotics, such as vancomycin, dalbavancin, telavancinand oritavancin, fluoroquinolones, tetracycline derivatives, hydrophobicderivatives of penicillins, cephalosporins, monobactams, carbapenems,penems and other betalactam antibiotics, novobiocin, pleuromutilins,folate synthesis inhibitors, deformylase inhibitors, and bacterialefflux pump inhibitors. A person skilled in the art of treatingGram-negative infections may easily recognize additional, clinicallyrelevant antibacterial agents that may be useful. Preferably saidantibacterial agents are selected from a group of hydrophobic ormoderately hydrophobic antibacterial agents against which the outermembrane of Gram-negative bacteria acts as an effective permeabilitybarrier.

The invention also includes the use of the present compounds orcombinations thereof to sensitize clinically important bacteria listedherein to the host defence mechanism complement (present in the freshhuman and animal serum) by subjecting said bacteria to the action ofsuch compounds during a clinical infection or a suspected infection. Thehost defence can be exerted, e.g., by the combined action of complementand polymorphonuclear leucocytes.

Those skilled in the art of medicine can readily optimize effectivedosages and administration regimens for the compounds according to thepresent invention as well as for the antibiotics in concurrentadministration, taking into account factors well known in the artincluding type of subject being dosed, age, weight, sex and medicalcondition of the subject, the route of administration, the renal andhepatic function of the subject, the desired effect, the particularcompound according to the present invention employed and the toleranceof the subject to it. Dosages of all antimicrobial agents should beadjusted in patients with renal impairment or hepatic insufficiency, dueto the reduced metabolism and/or excretion of the drugs in patients withthese conditions. Doses in children should also be reduced, generallyaccording to body weight.

The total daily dose of a derivative according to the present inventionadministered to a human or an animal can vary, for example, in amountsfrom 0.1 to 100 mg per kg body weight, preferably from 0.25 to 25 mg perkg body weight.

It will also be recognised by one skilled in the art that the optimalcourse of treatment, i.e., the number of doses given per day for adefined number of days, will be determined by the nature and extent ofthe condition being treated, the form, route and site of administration,and the particular patient being treated, and that such optimums can bedetermined by conventional techniques.

There is also provided a method for assaying a compound according to thepresent invention, said compound being a derivative of a naturalpolymyxin or octapeptin, wherein said derivative has a only 3 positivecharges and a terminal moiety (D) comprising 1 to 5 carbon atoms, incontrast to the naturally occurring compound from which it is derived,for the ability to sensitize a harmful Gram-negative to antibacterialagents and/or the complement present in the serum, said methodcomprising the step of contacting the bacterium with said derivative ofa natural polymyxin or octapeptin, and identifying derivativespossessing sensitizing activity towards said bacterium.

In a further aspect there is provided a method for developing novelantibiotics comprising the steps of

(a) providing a natural polymyxin or octapeptin compound, or aderivative thereof, carrying a total of 4 to 6 positive charges and aterminal moiety (D) comprising 1 to 5 carbon atoms,

(b) replacing from 1 to 3 residues carrying one or more positive chargeswith a residue not having a positive charge, or with a covalent bond,thereby generating a polymyxin derivative carrying 3 positive chargesand a terminal moiety (D) comprising 1 to 5 carbon atoms,

(c) assaying said polymyxin derivative for the ability to sensitizeGram-negative bacteria to antibacterial agent; and

(d) selecting compounds having the ability to sensitize Gram-negativebacteria to an antibacterial agent.

In one embodiment of the method of the invention, the terminal moietyR¹²—C(O), R¹²—(C═S), or R^(12′), wherein R¹² and R^(12′) are as definedabove. In another embodiment of the invention, the terminal moiety (D)is R(FA), which is an optionally substituted alkanoyl or alkyl residuehaving a total of 1 to 5 carbon atoms.

In a still further aspect of the invention there is provided a methodfor developing novel antibiotics comprising the steps of

(a) providing a natural polymyxin or octapeptin compound, or aderivative thereof, carrying a total of 4 or 5 positive charges, or atotal of 6 positive charges, as in deacylpolymyxins, and a terminalmoiety (D) comprising more than 5 carbon atoms,

(b) replacing from 1 to 3 residues carrying one or more positive chargeswith a residue not having a positive charge, or with a covalent bond,thereby generating a derivative of a polymyxin compound having 3positive charges,

(c) replacing a terminal moiety (D) comprising more than 5 carbon atomswith a terminal moiety (D) comprising 1 to 5 carbon atoms, therebygenerating a derivative of a polymyxin compound carrying 3 positivecharges and an a terminal moiety (D) comprising 1 to 5 carbon atoms,

(d) assaying said polymyxin derivative for the ability to sensitizeGram-negative bacteria to antibacterial agent; and

(e) selecting compounds having the ability to sensitize Gram-negativebacteria to an antibacterial agent.

In one embodiment of the method of the invention, the terminal moiety(D) is R¹²—C(═O), R¹²—(C=S), or R^(12′), wherein R¹² and R^(12′) are asdefined above. In another embodiment of the invention, the terminalmoiety (D) is R(FA), which is an optionally substituted alkanoyl oralkyl residue having a total of 1 to 5 carbon atoms.

In a still further aspect of the invention there is provided a methodfor developing novel antibiotics comprising the steps of

a) providing a polymyxin or octapeptin compound, or a derivativethereof, having a total of 4 to 6 positive charges and lacking theterminal moiety (D),

b) replacing from 1 to 3 residues carrying one or more positive chargeswith a residue not having a positive charge, or with a covalent bond,thereby generating a derivative of a polymyxin compound carrying 3positive charges;

c) introducing a terminal moiety (D) comprising 1 to 5 carbon atoms,thereby generating a polymyxin compound carrying 3 positive charges anda terminal moiety (D) comprising of 1 to 5 carbon atoms;

e) assaying said polymyxin derivative for the ability to sensitizeGram-negative bacteria to antibacterial agent; and

f) selecting compounds having the ability to sensitize Gram-negativebacteria to an antibacterial agent.

In one embodiment of the method of the invention, the terminal moiety(D) is R¹²—C(═O), R¹²—(C═S), or R¹²′, wherein R¹² and R^(12′) are asdefined above. In another embodiment of the invention, the terminalmoiety (D) is R(FA), which is an optionally substituted alkanoyl oralkyl residue having a total of 1 to 5 carbon atoms.

There is also provided in accordance with the present invention asemisynthetic polymyxin derivative obtainable by treating chemically orenzymatically naturally-occurring polymyxins or octapeptins,respectively, or those variants thereof which are manufactured bygenetically modified organisms. Chemical treatments include, but are notlimited to, those with acetanhydride, formic acid, hydrazine, and oxalicacid. Enzymatic treatments include, but are not limited to, with enzymessuch as polymyxin deacylase, ficin, papain, bromelain,subtilopeptidases, subtilisin, colistin hydrolase, and Nagarse.

Preferred compounds according to one embodiment are less cationic thannatural polymyxins or octapeptins, carry three (3) positive charges onlyand an R(FA) having 1 to 5 carbon atoms, and are:

(a) able to sensitize Gram-negative bacteria such as Escherichia coli,Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae,Citrobacter freundii, and Acinetobacter baumannii to antibiotics, and/or

(b) less toxic than clinically used polymyxins, as evidenced in in vivoanimal model, and/or

(c) less nephrotoxic than clinically used polymyxins, as evidenced in ananimal model and/or in an in vitro test that measures affinity of thecompounds to kidney structures, and/or

(d) able to cause less histamine liberation from the tissues thanclinically used polymyxins when administered topically or when inhaledas an aerosol, and/or

(e) pharmacokinetically more favorable, such as having a longer serumhalf life, increased renal clearance, increased urinary recovery and/orby being less inactivated by polyanionic tissue and pus constituentsthan clinically used polymyxins.

In a further embodiment, the compounds of the invention have one or moremore pharmacokinetically favorable properties as compared to nativepolymyxins or octapeptins (e.g., polymyxin A, polymyxin B,IL-polymyxin-B₁, polymyxin D, polymyxin E, polymyxin F, polymyxin M,polymyxin S, polymyxin T, circulin A, octapeptin A, octapeptin B,octapeptin C, or octapeptin ID). Examples of such pharmacokineticallyfavorable properties include a longer serum half life, increased renalclearance, or increased urinary recovery as compared to nativepolymyxins or octapeptins (such as polymyxin E).

In a further embodiment, the compounds of the invention may have agreater percent urinary recovery of an administered dose over 24 hoursthan polymyxin E (colistin). In another further embodiment, the urinaryrecovery, based on experiments with rats, is about 1% or greater, about5% or greater, about 10% or greater, about 15% or greater, about 20% orgreater, about 25% or greater, about 30% or greater, about 35% orgreater, about 40% or greater, about 45% or greater, or about 50% orgreater. In contrast, the urinary recovery of polymyxin E (colistin) wasdetermined to be about 0.18±0.14% of dose in 24 hours (Li et al., 2003),using the same dose and procedure.

In another further embodiment, the compounds of the invention may have agreater renal clearance than polymyxin E (colistin) when administeredusing the same route and dosing. In a further embodiment, the compoundsof the invention have a renal clearance, based on experiments with rats,greater than about 0.1 ml/min/kg, greater than about 0.5 ml/min/kg,greater than about 1.0 ml/min/kg, greater than about 2.0 ml/min/kg,greater than about 2.5 ml/min/kg, greater than about 3.0 ml/min/kg, orgreater than about 3.5 ml/min/kg. In another further embodiment, therenal clearance of the compounds of the invention may be at least 10times, at least 50 times, at least 100 times, at least 150 times, atleast 200 times, at least 250 times, or at least 300 times that ofpolymyxin E, when administered at the same dose and administrationroute.

In another further embodiment, the compounds of the invention may alsohave one or more pharmacokinetically favorable properties as compared tosimilar compounds with longer fatty acid tails (i.e., a terminal moietyor R(FA) having more than five carbon atoms). As shown in Example 8,NAB741 has increased renal clearance and increased urinary recovery ascompared to NAB739. The compounds are chemically identical except thatNAB741 has an acetyl terminal moiety and NAB739 has an octanoyl terminalmoiety.

Methods for synthesising compounds according to the present inventioninclude but are not limited to the following described below. For aspecific compound to be synthetised, an expert in the art is able tochoose the appropriate method.

1. Semisynthetic derivatives of polymyxins and octapeptins that carry anunchanged heptapeptide part and a modified acyl-aminoacyl side chain canbe made by the procedures described as follows:

Protection of the free amino groups in the starting material (polymyxinor octapeptin, or modifications thereof) by methods known to thoseskilled in the art. The protection can be achieved by the use ofresidues such as t-butoxycarbonyl (tBoc), fluorenylmethoxycarbonyl(Fmoc), benzyloxycarbonyl (CBZ, Z), allyloxycarbonyl (ALOC),3-pyridyl-N-oxide-methoxycarbonyl (as described in patent publication GB1323962), by using Schiff bases such as benzaldehyde by the methoddescribed in Japanese Patent publication 7115630/1971 or the like whichcan be removed by conventional conditions compatible with the nature ofthe product.

In conditions where the poor water solubility occasionally poses aproblem in the sub-sequent steps, the protection can be made by usingnegatively-charged blocking groups such as a sulfonic acid derivative ofFmoc or a carboxylic acid derivative of Fmoc, the method being describedin US patent publication 2006004185. The water solubility can also beenhanced by linking a suitable, removable, negatively charged, veryhydrophilic blocking group to the OH-group of threonine.

Thereafter, the compound is subjected to an enzymatic treatment withenzymes such as polymyxin deacylase, polymyxin hydrolase, papain, ficin,bromelain, subtilopeptidase, Nagarse or other enzymes that remove aterminal part of the side chain or even the entire side chain ofpolymyxin or octapeptin compounds. This treatment can optionally befollowed by the Edman degradation procedure. The resultant compoundlacks the entire side chain and consists of the cyclic heptapeptide partonly, but has a free N-terminal alpha amino group.

Alternatively, polymyxins and octapeptins that have amino groupsprotected by acid-stable groups such as benzyloxycarbonyl can be treatedby oxalic acid or formic acid to yield protected deacylderivatives, themethod being described by Kurihara et al. (1974). The procedure isfollowed by further enzyme treatment as above and/or by Edmandegradation to yield a heptapeptide.

Thereafter, a suitable residue is linked to the free alpha-aminoposition of the heptapeptide ring portion. The residue might contain anacyl or related residue (R(FA) having in total 1 to 5 carbon atoms),such as methyl, acetyl, propionyl, butanoyl, isobutanoyl, valeroyl, andisovaleroyl residue) as well as amino acid residues, up to three andpreferably two residues. For instance, one semisynthetic compound withan acyl group and two amino acid residues can be prepared by adding tothe above-described heptapeptide a synthetic N-(acyl)-threonyl-Dthreonylresidue. This can be achieved by conventional general techniques knownto those familiar with the art of organic chemistry, these techniquesincluding the use of N-hydroxysuccinimide-linked residues as describedin US 2006004185. In this particular synthesis the procedure may involvethe use of N-acetylthreonyl-Dserinyl-N-hydroxysuccinimide.

2. Acylated polymyxin nonapeptides carrying three (3) free amino groups.Polymyxin D possesses only four (4) positive charges and has DSer in theposition R3. The free amino groups of polymyxin D can be protected bythe means described above. This is followed by an enzymatic treatmentand an optional Edman degradation step, to yield a nonapeptide, whichcan then be acylated by acylisotiocyanate (by the method well-known to aperson skilled in the art and described in US 2006004185, by acylchloride (by the method well-known to a person skilled in the art anddescribed in Chihara et al. 1974), or by using residues linked toN-hydroxysuccinimide (by the method well-known to a person skilled inthe art and described in US 2006004185). Finally, the protective groupsare removed. The acylated polymyxin D nonapeptide carries only three (3)free amino groups, all in the heptapeptide ring portion.

In an analogous manner, acylated polymyxin S nonapeptide can be made. Itcarries only three (3) free amino groups.

3. Totally synthetic polymyxin and octapeptin derivatives can be made bythe very conventional methods known for those skilled in the art. Suchmethods include the liquid-phase synthesis procedures as well as thesolid-phase synthesis procedures described for instance by Sakura et al.(2004), Tsubery et al. (2000a, 2000b, 2002, 2005), and Ofek et al.(2004). The methods include e.g. the use of protecting agents such asFmoc, tBoc, and CBZ at strategic positions, as well as the cyclisationstep where DPPA (diphenyl phosphorazidate) or a mixture ofbenzotrizole-1-yl-oxy-tris-pyrrolidino-phophonium hexafluorophosphate(PyBop), N-hydroxybenzotriazole (HoBt), and N-methylmorpholine (NMM) isused. Fmoc derivatives of many non-trivial as well as D-amino acids arecommercially available. The amino terminus of the last amino acidresidue is left unprotected to enable direct reaction in the acylationprocedure with acids such as propionic acid, butyric acid, isobutyricacid, valeric acid, and isovaleric acid.

4. Acylation of the free N-terminal alpha-amino group of theintermediate compounds described above (paragraphs 1-3) can also beperformed by using anhydrides such as acetic anhydride (see Example 1),propionic anhydride, butyric anhydride, and valeric anhydride by usingconditions well-known to a person skilled in the art. N-formylation canbe performed by using p-nitrophenyl formate in N-methyl pyrrolidine andconditions well-known to a person skilled in the art. N-methylation canbe performed by using a mixture of formic acid and acetic anhydride indimethylformamide and conditions well-known to a person skilled in theart.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, patents, and patentapplications cited throughout this application are hereby incorporatedby reference. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for thepresent invention and embodiments thereof.

LIST OF REFERENCES

All references cited in the present application are hereby incorporatedby reference in their entirety.

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Chihara S, Ito A, Yahata M, Tobita T, Koyama Y. 1974. Chemicalsynthesis, isolation and characterization of a-N-fattyacyl colistinnonapeptide with special reference to the correlation betweenantimicrobial activity and carbon number of fattyacyl moiety. Agric BiolChem 38:521-529.

de Visser P C, Kriek N M A J, van Hooft P A V, Van Schepdael A, FilippovD V, van der Marel G A, Overkleeft H S, van Boom J H, Noort D. 2003.Solid-phase synthesis of polymyxin B₁ and analogues via a safety-catchapproach. J. Peptide Res. 61:298-306.

Kimura Y, Matsunaga H, Vaara M. 1992. Polymyxin B octapeptide andpolymyxin B heptapeptide are potent outer membranepermeability-increasing agents. J Antibiot 45:742-749.

Kurihara T, Takeda H, Ito H, Sato H, Shimizu M, Kurosawa A. 1974.Studies on the compounds related to colistin. IX. On the chemicaldeacylation of colistin and colistin derivatives. Yakugaku Zasshi94:1491-1494.

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EXAMPLES

The following examples illustrate certain embodiments of the presentinvention and should not be construed as limiting the scope of theinvention.

Example 1

Peptide Synthesis

Polymyxin derivatives (“NAB peptides” or “NAB compounds”) weresynthesized by conventional solid phase chemistry, using the standardFmoc protection strategy. The amino acid at the C-terminus iscommercially available as pre-attached to the solid phase and whencleaved off the resin with acid, yields a C-terminal carboxylic acid.

The strategy in the protection was to use three levels of orthogonalprotection, temporary Fmoc protection for the alpha amino functions,groups which are removed during the acid cleavage stage, andsemi-permanent protection to cover reactive side chain functions whilethe cyclisation reaction takes place. After cleavage of the peptide fromthe resin, the C-terminal carboxylic acid is reacted with an aminofunction on the side chain of one of the amino acids to form a cyclicpeptide. After the cyclisation step, the semi-permanent protectiongroups are removed to yield NAB peptide.

Accordingly, the alpha amino function of the amino acid was protected byfluorenyl-methoxycarbonyl (Fmoc) and Fmoc was removed by 20% piperidinein dimethylformamide (DMF) at every cycle. The amino acid that isinvolved with cyclisation, i.e., diaminobutyric acid, was protected byt-butoxycarbonyl (tBoc), an acid labile group which was removed at thecleavage step. The functional group of asparagine was protected bytritylation. All the other amino acids which have functional side chaingroups were protected by a group that is stable to the acid cleavagestage, i.e., benzyloxycarbonyl (Z). Amino acids phenylalanine andleucine naturally needed no side chain protection. The amino terminuswas not protected; this enabled direct reaction in the acylationprocedure.

The synthesis steps were performed in a commercial automatizedsynthesizer that employedO-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HCTU) as activator.

The acylation was performed by using a four-fold molar excess of eachamino acid or the fatty acid, four-fold molar excess of the activatorHCTU (see above), and an eight-fold molar excess of N-methyl morpholine.The reaction time was 30 min.

The amino acids were purchased already protected from standardsuppliers.

The peptide was removed from the resin by reaction with a solution of95% trifluoroacetic acid and 5% water for 2 hours at room temperature,to yield the partially protected product. The resulting peptide wasprecipitated with diethyl ether.

The cyclisation mixture used wasbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBop), N-hydroxybenzotri-azole (HoBt), and N-methyl morpholine (NMM)at the molar excess of 2, 2, and 4, respectively. The peptide wasdissolved in dimethylformamide, the cyclisation mix was added andallowed to react for 2 hours. The cyclised, protected peptide wasprecipitated by the addition of cold diethyl ether. Any residual PyBopwas removed by washing the peptide with water.

Acetylation for performed by using aceticanhydride—diisopropyl-ethylamine—DMF (1:1:18 by vol.).

The remaining side chain protection groups (Z) were removed by catalyticdehydrogenation. The peptide was dissolved in acetic acid-methanol—water(5:4:1), under an atmosphere of hydrogen and in the presence of apalladium charcoal catalyst.

The peptide was purified by reverse phase chromatography usingconventional gradients of acetonitrile:water:trifluoroacetic acid. Theproduct was dried by lyophilisation.

The yield was 10-20 mg representing approx. 10%-20% of the theoretical,calculated from the molar amount (approx. 100 micromoles) of the firstamino acyl residue bound to the resin.

The purity, as estimated by reversed phase HPLC was more than 90%.Within experimental error, the masses obtained were those expected fromthe theoretical values.

Example 2

Activity of the Compounds Against Escherichia coli and Pseudomonasaeruginosa

Peptides synthesized in Example 1, both carrying only three (3) positivecharges, were studied for their ability to sensitize E. coli to themodel antibiotic rifampin. This was tested employing LB agar (LB AgarLennox, Difco, BD, Sparks, Md., U.S.A) plates that contain increasingconcentrations (0.1 μg/ml, 0.3 μg/ml, 1 μg/ml) of rifampin(Sigma-Aldrich, St. Louis, Mo., U.S.A) as well as by using LB agarcontrol plates that did not contain rifampin.

The indicator organism E. coli IH3080 (K1:018) was an encapsulatedstrain originally isolated from a neonate suffering from meningitis(Vaara et al. 1984) and obtained from National Public Health Institute,Helsinki, Finland.

From an overnight-grown culture of IH3080 on LB agar, a suspension ofapprox. 10⁸ cells/ml was prepared in 0.9% NaCl. Aliquots of thissuspension were then pipetted on the agar plates and the plates weregently shaken to spread the suspension evenly on the entire surface ofthe plate. Thereafter, the unabsorbed part of the suspension was removedby using a Pasteur pipette. After the surface had dried, small wells(diameter, 2 mm) were drilled on the plates (five wells per plate) byusing a sterile sharp-edged narrow metal tube, single-use pipette tip,and vacuum suction. Alternatively, a swab was used to spread theinoculum. Samples (4 μl and 10 μl) of the peptide solution in 0.9% NaCl(at concentrations of 1 μg/ml and 0.1 μg/ml) were then pipetted to thewells and the sample fluids were allowed to absorb. Controls included0.9% NaCl solution without the compound to be tested. The plates werethen incubated for 18 h at 37° C. whereafter the diameters of growthinhibition zones around each well were measured; the diameter of thewell itself was not reduced. Finally, the diameters were converted tosurface areas of growth inhibition (in square mm).

Table 2 shows the activity of the novel compounds against E. coli IH3080as compared with that of control compounds. Even though both lacked thedirect antibacterial activity of NAB739, they sensitized at aconcentration of 4 μg/ml the target to a concentration of rifampin aslow as 0.1 μg/ml. Interestingly, NAB747 was directly antibacterialagainst P. aeruginosa ATCC 27853. In a well containing 10 μg of thepeptide, it caused a zone of inhibition with the surface area of 50 sqmm. At 4 μg, the corresponding value was 20 sq mm.

TABLE 2 Structure of the novel compounds and their activity against E.coli IH3080 Structure* Positive Compound Compound SEQ Peptide sequencecharges Direct Activity w. group name ID No. FA-part side chain cyclicpart total (cyclic) activity** rifampin*** Control Polymyxin B 1 MO (H)A XTX cy [XXfLXXT] 5 (3) 79 95 compounds Deacyl- 2 — +XTX cy [XXfLXXT] 6(3) 57 79 polymyxin B Deacylcolistin 3 — +XTX cy [XXlLXXT] 6 (3) 79 87Polymyxin B 4 — +TX cy [XXfLXXT] 5 (3) 0 20 nonapeptide Polymyxin B 5— + cy [XXfLXXT] 4 (4) 0 0 heptapeptide NAB 704 6 — +TZ cy [XXfLXXT] 4(3) 0 0 NAB 705 7 — +ZTZ cy [XXfLXXT] 4 (3) 0 0 NAB 701 8 — +TX cy[XXfLZZT] 3 (1) 0 0 NAB 702 9 — +TX cy [XXfLBBT] 3 (1) 0 0 NAB 703 11 —+TX cy [XXfLJJT] 3 (1) 0 0 Octanoyl 12 OA — cy [XXfLXXT] 3 (3) 0 0 PMBHNAB 736 13 DA — [XXfLXXT ] 3 (3) 0 113 NAB 739 14 OA Ts cy [XXfLXXT] 3(3) 133 177 NAB 740 15 DA Ts cy [XXfLXXT] 3 (3) 95 95 NAB 7061 16 OA TZcy [XXfLXXT] 3 (3) 0 113 Novel NAB 741 29 Ac Ts cy [XXfLXXT] 3 (3) 0 95compounds NAB 745 28 Ac Tn cy [XXfLXXT] 3 (3) 0 50 NAB 747 10 Me Ts cy[XXfLXXT] 4 (3) 0 28 *One-letter codes for amino acyl residues: F, Phe;L, Leu; N, Asn; S, Ser; T, Thr; X Dab; Z, Abu; B, N-gammaformyl-Dab; J,N-gamma-acetyl-Dab. Small letters indicate residues that are inD-configuration. + indicates the positive charge of the alpha-aminogroup in the free N-terminus of the peptide. Abbreviations: MO(H)A, themixture of 6-methyloctanoyl, 6-methylheptanoyl and related fatty acidresidues occurring in polymyxin B; OA, octanoyl; DA, decanoyl; Ac,acetyl; Me, methyl. **Antibacterial activity measured as the growthinhibition (in square millimeters) around a well containing 4 microgramsof the compound on LB plates. ***Antibacterial activity measured as thegrowth inhibition (in square millimeters) around a well containing 4micrograms of the compound on a LB plate containing rifampin (0.1micrograms/ml).

Example 3

NAB741 Sensitizes E. coli, Klebsiella pneumoniae, and Enterobactercloacae to a Broad Range of Antibacterial Agents

The minimum inhibitory concentrations (MIC) of a representative set ofclinically used antimicrobial agents were determined for two strains ofE. coli (ATCC25922 and IH3080), K. pneumoniae ATCC13883, and E. cloacaeATCC23355 by using Mueller-Hinton agar medium (product no LabO39; LabMLtd., Bury, Lancs, U.K.) in the presence of NAB741 (4 μg/ml) as well asin its absence. MICs were determined by using E-strips (Biodisk Ltd.,Solna, Sweden) according to the manufacturer's instructions. The NAB741concentration used did not itself inhibit the growth of the targetbacteria. The MIC of NAB741 for all these strains was >16 μg/ml.

The results are shown in Table 3. NAB741 at a concentration of 4 μg/mlwas able to sensitize the tested strains to rifampin by a factor rangingfrom >64 to >2000. Sensitization factor is defined as the ratio of theMIC of an antibiotic in the absence of NAB741 to that in the presence of4 μg/ml of NAB741. Extremely high sensitization factors were observedalso to clarithromycin (24-340), mupirocin (8-192), azithromycin(16-32), for some of the strains to fusidic acid (128-170), and for E.cloacae to vancomycin (170). All these antibacterial agents are notablyhydrophobic or large (vancomycin) and are known to be excluded by theintact OM of Gram-negative bacteria but penetrate the damaged OM.

TABLE 3 Sensitization factors* to selected antibacterial agents at NAB741 concentration of 4 μg/ml E. coli K. pneum. E. cloacae ATCC E. coliATCC ATCC 25922 IH 3080 13883 23355 Rifampin 750 250 >64 >2000Clarithromycin 340 96 24 96 Mupirocin 128 64 8 190 Azithromycin 24 32 3216 Fusidic acid 170 130 >5 >130 Vancomycin >16 16 >2 170 *Sensitizationfactor is the ratio of the MIC of the antibiotic in the absence of NAB741 to that in the presence of 4 μg/ml of NAB 741

Example 4

Susceptibility of Seven Different Strains of Gram-Negative Bacteria toRifampin and Clarithromycin in the Presence of NAB741 (4 μg/ml)

The minimum inhibitory concentrations (MIC) of rifampin andclarithromycin for a representative set of different strains ofclinically relevant Gram-negative bacteria were determined by the E-testmethod as in Example 3 and by using Mueller-Hinton agar with or withoutNAB741 (4 μg/ml). This concentration of NAB741 did not itself inhibitthe growth of the target bacteria. Five of the strains originated fromATCC. Acinetobacter baumannii F264 was purchased from Mobidiag Ltd.,Helsinki, Finland. The source of E. coli IH3080 has been given inExample 2.

The results are shown in Table 4. It shows that NAB 741 is remarkablyactive even against Acinetobacter baumannii.

TABLE 4 The ability of NAB 741 to sensitize Gram-negative bacteria tomodel antibiotics (rifampin and clarithromycin) MIC (μg/ml) of MIC(μg/ml) of rifampin in the clarithromycin in presence of Sensitizationthe presence of Sensitization 4 μg/ml factor* 4 μg/ml factor** Bacterialstrain of NAB 741* to rifampin of NAB 741 to clarithromycin E. coliATCC25922 0.016 750 0.125 340 E. coli IH3080 0.047 250 0.125 96 K.pneumoniae 0.5 >64 1 24 ATCC13883 E. cloacae ATCC23355 0.016 2000 0.5 96Ac. baumannii 0.19 16 0.5 32 ATCC19606 Ac. baumannii F264 0.125 64 0.532 P. aeruginosa 16 2 64 2 ATCC27853 *The ratio of rifampin MIC in theabsence of NAB 741 to that in the presence of NAB 741 (4 μg/ml). **Theratio of clarithromycin MIC in the absence of NAB 741 to that in thepresence of NAB 741 (4 μg/ml).

Example 5

NAB741 Sensitizes a Meropenem-Resistant strain of Acinetobacterbaumannii to Meropenem

The minimum inhibitory concentrations (MIC) of meropenem for two strainsof A. baumannii were determined by the E-test method as in Example 4 andby using Mueller-Hinton agar with or without NAB741 (4 μg/ml). Thisconcentration of NAB741 did not itself inhibit the growth of the targetbacteria. Sensitization factor was defined as in Example 4. The resultsare shown in Table 5. NAB7061 sensitized the meropenem-resistant strainF264 to meropemen by a factor >4.

TABLE 5 Sensitization of the meropenem-resistant strain of Acinetobacterbaumannii to meropenem in the presence of NAB 741 (4 μg/ml) MIC (μg/ml)of meropenem at the indicated concn (μg/ml) of NAB 741 Strain 0 4 A.baumannii ATCC19606 0.75 0.5 A. baumannii F264 >32 8

Example 6

NAB741 Sensitizes E. coli to the Complement in Fresh Normal Serum

The ability of NAB741 to sensitize encapsulated, smooth strain of E.coli to the bactericidal action of normal guinea pig serum (GPS) wasstudied by the method described by Vaara et al. (1984). E. coli IH3080(018,K1) was grown in LB broth (LB broth Lennox, Difco, BD, Sparks, Md.,U.S.A) at 37° C. in a rotary shaker into early logarithmic growth phase,washed with PBS (phosphate-buffered saline, 8.0 g of NaCl, 0.2 g of KCl,1.44 g of Na₂HPO₄ x2H₂O and 0.2 g of KH₂PO₄ per liter) and resuspendedin PBS, to approx. 10⁹ cells/ml). GPS was used as complement source. Itwas stored at −70° C. before use. To inactive the complement, serum wasincubated at 56° C. for 30 min.

The experimental procedure was as follows. 10% GPS in PBS was inoculatedwith approx. 500 CFU (colony forming units) of bacteria per ml andpipetted in 0.2 ml aliquots into wells of microtiter plates. The wellsalready contained increasing amounts of NAB7061 in 0.020 ml of 0.9%NaCl. The plate was incubated at 37° C. for 2 h whereafter each well wasemptied onto LB plates. The plates were incubated overnight at 37° C.and the developed colonies were counted.

The results are shown in Table 6. NAB741 itself did not significantlyreduce CFU count in the absence of GPS or in the presence ofheat-inactivated 10% GPS. However, as low a concentration of NAB741 as 2μg/ml was sufficient to reduce CFU count by a factor of approx. 100 inthe presence 10% fresh GPS. Accordingly, NAB741 acts synergisticallywith the bactericidal complement machinery present in fresh serum, asdoes PMBN, the agent well known to have this property.

TABLE 6 The synergistic bactericidal activity of NAB741 and 10% guineapig serum (GPS) against E. coli IH3080 (O18:K1)* Concentration of NAB741(μg/ml) 0 1 2 4 none (PBS) 100 71 86 58 10% GPS >200 5 7 0 10% GPS, heatinactivated >200 >200 >200 125 *measured as % survival after 2-hourtreatment at 37° C.

Example 7

Preparation and Biological Activity of NAB 739 Methanesulfonate SodiumSalt

NAB 739 acetate (100 mg) was dissolved in water (2 ml) and neutralformaldehyde solution (400 microliters of 30% aqueous formaldehyde[brought to pH 7.2 with 1N Na-HCO3]) was added. Then, 1N NaHCO3 solution(2 ml) was added, and the precipitated NAB 739 formaldehyde derivativewas filtered and washed with water. The moist solid was suspended inwater (5 ml), and sodium metabisulphite (100 mg) was added. A clearsolution was obtained after a few minutes and was freeze-dried. Theflocculent white solid was extracted with warm acetone (7.5 ml) anddried in vacuo. The yield was 56 mg. Analysis of the product by ESI massspectrometry revealed a predominant peak with the molecular mass of1075.3 indicating that most of the derivative was sulfomethylated ateach of the three Dab residues of the NAB 739 compound. A minor peakrepresenting the NAB 739 blocked randomly at two of the three Dabresidues was also visible.

For the measurement of the antibacterial activity of aqueous solutionsof NAB 739 methanesulfonate sodium, three different solutions weremade: 1) A solution (1 mg/ml) made in 0.9% NaCl immediately prior to theexperiment, 2) a solution (1 mg/ml) made in 0.9% NaCl 24 h prior to theexperiment and kept at 37° C., 3) a solution (1 mg/ml) made in 0.9% NaCl48 h prior to the experiment and kept at 37° C. A freshly made solutionof NAB 739 acetate served as the control compound.

TABLE 7 The bactericidal activity of NAB 739 methanesulfonate ascompared with that of NAB 739 against E. coli IH3080* Compound Solutionage 0 1 2 4 NAB 739 MS** fresh 100 70 62 16 NAB 739 MS** 24 h 55 34 6NAB 739 MS** 48 h 35 11 0 NAB 739 fresh 21 2 0 *measured as % survivalafter 2-hour treatment at 37 degrees centigrade. **MS, methanesulfonate

The test bacterium was E. coli IH3080. It was grown in LB broth (LBbroth Lennox, Difco, BD, Sparks, Md., U.S.A.) at 37° C. in a rotaryshaker into early logarithmic growth phase, washed with PBS, andresuspended in PBS to approx. 10e9 cells/ml. PBS was inoculated withapprox. 500 CFU (colony forming units) of bacteria per ml and pipettedin 0.2 ml aliquots into wells of a microtiter plate. The plates alreadycontained increasing concentrations of NAB 739 methanesulfonate or thecontrol compound in 0.020 ml of 0.9% NaCl. The plate was incubated at37° C. for 1 h whereafter each well was emptied onto LB plates. Theplate was incubated overnight at 37° C. and the developed colonies werecounted.

The results are shown in Table 7. The fresh solution of NAB 739methanesulfonate was much less antibacterial than the control compoundNAB 739. Keeping the NAB 739 methanesulfonate solution at 37° C. for 24h prior to use slightly increased the activity whereas keeping for 48 hresulted in activity almost equal to that observed with the controlcompound. These results indicate that NAB 739 methanesulfonate, inanalogy with colistin methanesulfonate, slowly decomposes in aqueoussolutions to yield antibacterially more active substances, i.e., lesssulfomethylated substances and eventually free NAB 739.

Similarly, methanesulfonate derivatives of NAB 741, NAB 745, NAB 747 andother compounds described as herein are prepared. These prodrugsdecompose in vivo to yield compounds which possess the ability tosensitize target bacteria to other antibacterial agents and serumcomplements.

Example 8

Comparison of Basic Pharmakokinetic Properties of NAB 741 and NAB 739

The studies were principally performed by using the methods described byLi et al. (2003, 2004). Each rat (n=4 for both compounds,Sprague-Dawley, male) was anaesthetized using isoflurane, and apolyethylene cannula was inserted into the jugular vein. Each rat wasplaced into a metabolic cage and allowed to recover from the procedureovernight. The test compound (acetate, 1 mg/kg) was administered as abolus (in 200 μI sterile 0.9% saline) through the cannula, followed bywashing with 0.8 ml of saline. Nine blood samples (0, 10, 20, 30, 60,90, 120, 180, and 240 min), each 200 μl, were manually collected throughthe cannula. When collecting samples, the first 100 μl of blood waswithdrawn and kept in the syringe. After collecting the actual samplewith another syringe, the content of the first syringe was returned tothe rat together with 400 μl of heparinized saline. Blood samples werecentrifuged to obtain plasma. Urine samples were collected in 0-4 h, 4-6h, and 6-24 h intervals. Plasma and urine samples were stored at −80° C.

The samples were analyzed using liquid chromatography and massspectrometry with electrospray ionization interface (LC/electrosprayionization MS). To a 100-μl sample, 10 μl of internal standard (NAB 739,80 μg/ml) and 200 μl (plasma samples) or 100 μl (urine samples) ofacetonitrile was added, the mixture was vortex-mixed for 1 min, andcentrifuged at 10.000 g for 10 min. The chromatography employed the HPLCC18 column (50×2 mm), 0.1% formic acid as the solvent A, 0.1%acetonitrile as the solvent B, flow rate of 0.2 ml/min, and thefollowing gradient: 5%-30% B in 6 min, 30%-90% B in 0.5 min, 90% B heldfor 2.5 min, 90%-5% B in 1 min. The eluent between 5.90-7.00 min and9.00-10.1 min was directed to the MS system using a switching valve. Thepositive protonated molecular ions of NAB 741 at m/z 496.7 and 331.4 andof NAB 739 at m/z=538.8 and 359.6 and were monitored. NAB 741 was elutedat 6.65±0.05 min and NAB739 was eluted at 9.45±0.05 min.Non-compartmental analysis of the compounds in plasma was performedusing WinNonlin software (version 4.0, Mountain View, Calif., USA), withthe model of NA201 (i.v. bolus input for plasma data).

The basic pharmacokinetic parameters determined for NAB 741, were asfollows: half-life (min), 32.7±2.41; volume of distribution (ml/kg),243±24.0; clearance (ml/min/kg), 7.39±0.85; urinary recovery (% of dosein 24 h), 50.9±13.6; and renal clearance (ml/min/kg), 3.78±1.11.

The basic pharmacokinetic parameters determined for NAB 739, a compoundotherwise identical to NAB 741 but having octanoyl residue instead ofacetyl residue as its terminal moiety, were as follows: half-life (min),69.0±21.9; volume of distribution (ml/kg), 222±20.5; clearance(ml/min/kg), 2.63±0.54; urinary recovery (% of dose in 24 h), 19.4±7.38;and renal clearance (ml/min/kg), 0.53±0.30.

The corresponding parameters for colistin, as determined by Li et al.(2003) by using an identical dosing and administration procedure, arethe following: half-life (min), 74.6±13.2; volume of distribution(ml/kg), 496±60; clearance (ml/min/kg), 5.2±0.4; urinary recovery (% ofdose in 24 h), 0.18±0.14; and renal clearance (ml/min/kg), 0.010±0.008.

The invention claimed is:
 1. A polymyxin derivative of formula (I):

wherein: A is a polymyxin ring moiety having the following formula:

wherein R4 of the polymyxin ring moiety is an amino acid residueselected from the group consisting of lysine, hydroxylysine, ornithine,glutamate, aspartate, an α,γ-diamino-n-butyryl residue, diaminopropionicacid, threonine, cysteine and serine; R6 is an amino acid residueselected from the group consisting of D-phenylalanine, D-leucine andD-tryptophan; R7 is an amino acid residue selected from the groupconsisting of leucine, threonine, phenylalanine, isoleucine, serine,alanine, valine, norvaline, and tryptophan; R10 is an amino acid residueselected from the group consisting of leucine, threonine, serine,O-acetyl-threonine, O-propionyl-threonine and O-butyryl-threonine; andwherein R5, R8 and R9 are amino acid residues independently selectedfrom the group consisting of an α,γ-diamino-n-butyryl residue, lysine,2-amino-4-guanido butyric acid, an α-aminobutyryl residue, andthreonine; D is R¹²—(C═O); R¹²—SO₂—; R¹²—(C═NH)—; R¹²—NH—(C═S)—;R¹²—NH—(C═O)—; R¹²—NH—(C═NH)—;R¹²—O—(C═S)—; R¹²—O—(C═O); R¹²—P(O)O H—;R¹²—(C═S); or R^(12′), wherein R¹² and R^(12′) are alkyl, cycloalkyl,alkenyl, alkynyl, aryl, or aryl alkyl, and wherein D has no more than 1to 5 carbon atoms; m¹, m², and m³ are each independently 0 or 1; Q¹, Q²,and Q³ are each independently CH₂, C═O, or C═S; W¹, W², and W³ are eachindependently NR⁴, O, or S; R^(1′), R^(2′), and R^(3′)are eachindependently side chains of natural or unnatural amino acids, alkyl,alkenyl, alkyl, arylalkyl, aryl, alkoxy, alkoxycarbonyl,aryloxycarbonyl, alkylamino, or alkynyl; and R⁴ is hydrogen or alkyl,and pharmaceutically acceptable prodrugs and salts thereof wherein saidderivative has three positive charges at physiological pH; and providedthat at least one of m¹, m², and m³ are
 1. 2. The polymyxin derivativeof claim 1, wherein m¹ is
 0. 3. The polymyxin derivative of claim 1,wherein m² and m³ are each
 1. 4. The polymyxin derivative of claim 1,wherein Q² and Q³ are each C═O.
 5. The polymyxin derivative of claim 1,wherein W² and W³ are each NH.
 6. The polymyxin derivative of claim 1,wherein R^(1′), R^(2′), and R^(3′)comprise one or more hydroxyl,carbamyl, amidyl, carboxylate, thiol, sulfate, sulfonyl, or phosphategroups.
 7. The polymyxin derivative of claim 1, wherein A is a polymyxinring moiety selected from that of polymyxin A, polymyxin B,IL-polymyxin-B₁, polymyxin D, polymyxin E, polymyxin F, polymyxin M,polymyxin S, polymyxin T, circulin A, octapeptin A, octapeptin B,octapeptin C, or octapeptin D.
 8. The polymyxin derivative of claim 1,wherein the derivative is of the formula (II):

wherein: A is a polymyxin ring moiety having the following formula:

wherein R4 of the polymyxin ring moiety is an amino acid residueselected from the group consisting of lysine, hydroxylysine, ornithine,glutamate, aspartate, an α,γ-diamino-n-butyryl residue, diaminopropionicacid, threonine, cysteine and serine; R6 is an amino acid residueselected from the group consisting of D-phenylalanine, D-leucine andD-tryptophan; R7 is an amino acid residue selected from the groupconsisting of leucine, threonine, phenylalanine, isoleucine, serine,alanine, valine, norvaline, and tryptophan; R10 is an amino acid residueselected from the group consisting of leucine, threonine, serine,O-acetyl-threonine, O-propionyl-threonine and O-butyryl-threonine; andwherein R5, R8 and R9 are amino acid residues independently selectedfrom the group consisting of an α,γ-diamino-n-butyryl residue, lysine,2-amino-4-guanido butyric acid, an α-aminobutyryl residue, andthreonine; D is R¹²—C(═O), R¹²—C(═S), or R^(12′); m¹, m², and m³ areeach independently 0 or 1, provided that at least one of m¹, m², and m³are 1; R^(1′), R^(2′), and R^(3′)are each independently side chains ofnatural or unnatural amino acids, alkyl, alkenyl, arylalkyl, aryl,alkoxy, alkoxycarbonyl, aryloxycarbonyl, alkylamino, or alkynyl ; andR¹² is C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl, R^(12′)is C₁-C₅alkyl, C₂-C₅ alkenyl, or C₂-C₅ alkynyl, and pharmaceutically acceptableprodrugs and salts thereof, wherein said derivative has three positivecharges at physiological pH.
 9. The polymyxin derivative of claim 8,wherein m¹ is
 0. 10. The polymyxin derivative of claim 8, wherein m² andm³ are each
 1. 11. The polymyxin derivative of claim 8, wherein R¹² isC₁-C₄ alkyl.
 12. The polymyxin derivative of claim 11, wherein D isacetyl, propionyl, butanoyl, or pentanoyl.
 13. The polymyxin derivativeof claim 1, wherein the derivative is of the formula (III):

wherein: A is a polymyxin B or polymyxin E ring moiety having thefollowing formula:

wherein R4 is diaminobutyric acid, R5 is diaminobutyric acid, R6 isD-phenylalanine, R7 is leucine, R8 is diaminobutyric acid, R9 isdiaminobutyric acid and R10 is threonine; or R4 is diaminobutyric acid,R5 is diaminobutyric acid, R6 is D-leucine, R7 is leucine, R8 isdiaminobutyric acid, R9 is diaminobutyric acid and R10 is threonine; Dis R¹²—C(═O) or R¹²—C(═S); m¹ is 0 or 1; R^(1′), R^(2′), and R^(3′)areeach independently side chains of natural or unnatural amino acids,alkyl, alkenyl, arylalkyl, aryl, alkoxy, alkoxycarbonyl,aryloxycarbonyl, alkylamino, or alkynyl, wherein at least one ofR^(2′)and R^(3′)comprise a carbamyl, hydroxyl or carboxylate group; R¹²is C₁-C₄ alkyl; and pharmaceutically acceptable prodrugs and saltsthereof, wherein said derivative has three positive charges atphysiological pH.
 14. The polymyxin derivative of claim 13, wherein m¹is
 0. 15. The polymyxin derivative of claim 13, wherein R^(2′)andR^(3′)each comprise a carbamyl, hydroxyl or carboxylate group.
 16. Thepolymyxin derivative of claim 13, wherein D is acetyl, propionyl,butanoyl, or pentanoyl.
 17. The polymyxin derivative of claim 1, whereinthe derivative is of the formula (IV):

wherein: A is a polymyxin B or polymyxin E ring moiety having thefollowing formula:

wherein R4 is diaminobutyric acid, R5 is diaminobutyric acid, R6 isD-phenylalanine, R7 is leucine, R8 is diaminobutyric acid, R9 isdiaminobutyric acid and R10 is threonine; or R4 is diaminobutyric acid,R5 is diaminobutyric acid, R6 is D-leucine, R7 is leucine, R8 isdiaminobutyric acid, R9 is diaminobutyric acid and R10 is threonine; m¹is 0 or 1; L¹, L² and L³ are each independently C₁-C₃ alkyl or acovalent bond; M¹, M² and M³ are each independently H, C(═O)NH₂,C(═O)OH, or —OH; R¹² is C₁-C₄ alkyl, and pharmaceutically acceptableprodrugs and salts thereof, wherein said derivative has three positivecharges at physiological pH.
 18. The polymyxin derivative of claim 17,wherein m¹ is
 0. 19. The polymyxin derivative of claim 17, wherein L² is—CH(CH₃)— and M² is OH; L² is —CH₂— and M² is H; or L² is —CH₂— and M²is OH.
 20. The polymyxin derivative of claim 17, wherein L³ is —CH₂— andM³ is OH or ; L³ is —CH₂—CH₂— and M³ is C(═O)NH₂.
 21. The polymyxinderivative of claim 1, wherein the derivative is of the general formula(V),

wherein R4 is an amino acid residue selected from the group consistingof lysine, hydroxylysine, ornithine, glutamate, aspartate, anα,γ-diamino-n-butyryl residue, diaminopropionic acid, threonine,cysteine and serine; R6 is an amino acid residue selected from the groupconsisting of D-phenylalanine, D-leucine and D-tryptophan; R7 is anamino acid residue selected from the group consisting of leucine,threonine, phenylalanine, isoleucine, serine, alanine, valine,norvaline, and tryptophan; R10 is an amino acid residue selected fromthe group consisting of leucine, threonine, serine, O-acetyl-threonine,O-propionyl-threonine and O-butyryl-threonine; wherein R5, R8 and R9 areamino acid residues independently selected from the group consisting ofan α,γ-diamino-n-butyryl residue, lysine, 2-amino-4-guanido butyricacid, an α-aminobutyryl residue, and threonine; wherein R1 is optional;wherein R1, R2 and R3 are independently selected from a group consistingof alanine, aminobutyric acid, asparagine, aspartic acid, glutamic acid,glutamine, serine, or threonine, in either an L or D configuration; andwherein R(FA) is an optionally substituted alkanoyl or alkyl residuehaving no more than 1 to 5 carbon atoms; or pharmaceutically acceptableprodrugs and salts thereof, wherein said derivative has three positivecharges at physiological pH.
 22. The derivative according to claim 21,wherein R(FA) is a residue having no more than 1 to 3 carbon atoms. 23.The derivative according to any one of claim 1 or 21, wherein saidderivative is a prodrug comprising one or more positive charge maskingmoieties.
 24. The derivative of any one of claim 1 or 21, wherein saidderivative has one or more pharmacokinetically favorable properties ascompared to native polymyxins, octapeptins, or polymyxin derivativeswith terminal moieties with more than five carbon atoms.
 25. Thederivative of claim 24, wherein said pharmacokinetically favorableproperty is a longer serum half life, increased renal clearance, orincreased urinary recovery as compared to native polymyxins,octapeptins, or polymyxin derivatives with terminal moieties with morethan five carbon atoms.
 26. A combination product comprising two or moreof the derivatives according to any of claim 1 or
 21. 27. Apharmaceutical composition comprising at least one derivative accordingto any one of claim 1 or 21, and at least one pharmaceuticallyacceptable carrier and/or excipient.
 28. The pharmaceutical compositionaccording to claim 27, further comprising a second antibacterial agent.29. A method for sensitizing Gram-negative bacteria to an anti-bacterialagent, comprising administering, simultaneously or sequentially in anyorder, a therapeutically effective amount of said antibacterial agentand a derivative according to any one of claim 1 or
 21. 30. A method fortreating a Gram-negative bacterial infection in a subject, comprisingadministering an effective amount of a derivative of any one of claim 1or 21 in combination with a second antibacterial agent, such that thebacterial infection is treated.
 31. The polymyxin derivative of claim21, wherein R4-R10 together represent a polymyxin ring moiety selectedfrom the group consisting of polymyxin A, polymyxin B, IL-polymyxin-B1,polymyxin D, polymyxin E, polymyxin F, polymyxin M, polymyxin S,polymyxin T, circulin A, octapeptin A, octapeptin B, octapeptin C, andoctapeptin D.
 32. The polymyxin derivative of claim 21, wherein R1 isabsent.
 33. The polymyxin derivative of claim 21, wherein R2 is L-serineor L-threonine.
 34. The polymyxin derivative of claim 21, wherein R3 isD-asparagine, L-serine, D-serine, L-threonine or D-threonine.
 35. Thepolymyxin of claim 21, wherein R(FA) is an optionally substitutedalkanoyl residue having no more than 1-5 carbon atoms.